Difference between revisions of "Team:USTC/Description"

Latest revision as of 16:00, 14 October 2015

Overview
CACCI
SPRING
ROSE

NDM: Nanomachine Detecting Microbiotics

Abusing antibiotics has caused severe antibiotics contamination and resistance issues worldwide. Therefore, we USTC develop a device NDM with an optical interference path, recognition program based on RasberryPi and Arduino to detect antibiotics in natural water bodies. In NDM, there is a system for measurement: CACCI which contains chemotaxis modified bacterial adhered to a polymer membrane. Deformation caused by CACCI motility can be recognized with optical interference and the interference pattern recognition program inside NDM will analyze the deformation, thus antibiotic situation in sample can be accurately told.

What is Antibiotics?

Antibiotics, or microbiotics, in narrow sense, are a various type of artificially constructed or naturally existing chemicals containing the capability of killing bacteria or inhibiting their growth, which are implemented widespread around the world. Patients who got bacterial or protozoan infection from surgical wound like war field, empyrosis and previously regarding severe diseases such as general cold, tuberculosis(TB), sexual transmitted diseases(STD), dysentery, anthrax, even cancer and so forth are able to cure treating with antibiotic substances. Since the discovery of penicillin by British biologists Alexander Flaming in 1929, the antibiotic family has been enlarged a lot. With properly administration of antibiotics, millions of people were saved in evidently alleviated symptom. There was a time when antibiotics, along with oil source for automobile, was indispensable for human beings.

Antibiotic substances containing several mechanism, rendering them to kill and inhibit bacterial growth in their own ways. Those mechanisms including:

Inhibit the synthesis of cell wall.
Influence the function of cell membrane.
Inhibit the biosynthesis of nucleic acid.
Inhibit the biosynthesis of proteins.

Classes categorization based on chemical structure including:

Beta-lactam
Aminoglycoside
Amide alcohol
Macrolide
Polypeptide
Tetracycline
Sulfonamides
Quinolones
Polymyxin
Trimethoprim
Nitrofuran etc.

Ordered formal course of treatment, these antibiotic would effectively treat
gonorrhea, MRSA and pseudomonal infections respectively.

However, treating with microbiotics may be associated with a rang of side effects, from some mild including gastrointestinal upset, diarrhea to severe like hearing loss, kidney damage. Some speculation from scientists postulated that exposition of high concentration utilization of antibiotics alter the host microbiota, especially the family of the probiotics, and this has been associated with chronic diseases.

The production of antibiotics in pharmaceutical companies explosively expanded these years. Artificial synthesis of sulfonamides, quinolones and oxazolidinones enrich the categories of antibiotics.

Antimicrobial Resistance and Abuses

As the matter of fact, microbiotics, the great saver of our life, is now putting us in potentially tremendous disaster both environmentally and physiologically.

Emergence of antimicrobial resistance is theoretically predictable by evolutionary potential. The antibiotic treatment may select for bacterial strains physiologically improved of their cell bodies or genetically enhanced capacity of mutagenesis and gene horizontal transfer like conjugation.

Because of such horizontal genetic exchange, nowadays, efficacy loss may attribute to the emergence of many antimicrobrial-resistent strains. For instance, tuberculosis(TB) in China becomes more difficult with administration with previous antibiotic substances owing to elevating level of antimicrobrial resistance, which raised great challenge on Chinese public health. Besides, the discovery of NDM-1(New Delhi Metallo-beta-lactamase) in Klebsiella pneumoniae from a Swedish patient of Indian origin in 2008 once may contribute to severe disaster on that broad of beta-lactam antibiotics.

Though, antimicrobial resistance can be transferred through evolutionary theory, main contribution of increasing antibiotic resistance comes from individuals' inappropriate antibiotics treatment, overuse of antibiotics without prescription from professional doctor, instead from self-prescription.

Demonstrably, attribution of antibiotic resistance not only from clinically, but from agriculture and animal husbandry. In China, the overuse of antibiotics for animal is the same serious as the clinical therapy. In farms or hospitals, solution containing antibiotics directly emits to river, which potentially strongly influence the ecological equilibrium of bactierial colonies and sidelong threaten public health.

Antibiotics Production and Contamination in China

Because individuals neglecting antibiotic emission, rivers in China now encounter extremely antibiotic contamination.

According to the latest research from Chinese Academy of Science published on ACS Environmental Science and Technology, China used 162,000 tons of antibiotic substances for human(48%) and animal(52%) in 2013, which is tenfold usage of USA and antibiotic substances in high concentration can be detected from many river basins that may potentially harmful to public health and environment. In their professional evaluation, antibiotics can be detected in all rivers in China in different degree.

Among these rivers, the Pearl River Basin in South China and Hai River Basin in North China received the highest antibiotics emission. More than 36 common antibiotic substances were discovered in these rivers, one of them, for example, in the Pearl River Basin, amoxicillin was found at 3384 ng/L and fluprofen at 2867 ng/L. Though lack of standard ranking on antibiotic contamination, according to environmental scientists from CAS, antibiotic substances concentration more than 1000 ng/L is relatively extremely high, based on Europe Union Standard.

Now, many technology including ELISA, Delvotest, HPLC-MS/MS, CE and Quantum Dot are able to detect antibiotic concentration in river sample, however much of their have much deficiencies, including:

Not portable, many big and automatic equipment are indispensable for concentration detection.
Complicated Routine, which requires trained personnel to conduct experiment. However, facing that large scale contamination, complicated routine and trained personnel cannot be satisfied at all districts, so do it in many developing and undeveloped countries and districts.
Recent technology is still not sensitive enough and sometimes time consuming.

Hope with our project, antibiotics issues can be solved in an accelerated way.

NDM: Nanomachine Detecting Microbiotics
What is Antibiotics?
Antimicrobial Resistance and Abuses
Antibiotics Production and Contamination in China

Who is CACCI?

CACCI, called the same as Kathy, is a kind of constructed bacteria for Characterization of Antibiotic Concentration based on Chemotaxis and Interference.

Along with ROSE, CACCI embedded in SPRING will be used to detect antibiotic. Although, the construction of ROSE will specifically tell the concentration of different antibiotics in the river samples, its feature is also deficiency: that is such strong specificity may hurt extensive measurement. Besides, using traditional genetically modified circuits to express report is still time-consuming, comparing with general physical or chemical measurement approaches.

Consequently, we originally established CACCI based on chemotaxis and interference to make a larger level of antibiotic molecule detection, even a detection for other chemical molecules. So, the mechanism and construction of CACCI can be divided into the following schedules:

Chemotaxis Modification: to increase the bacterial mobility, modification based on E. coli chemotaxis related to the parts cheZ will be introduced into bacteria.
Permeability Improvement: Because antibiotic substances contamination occurs in very low concentration, such as ~2000ng/L or 2ug/L that is much lower than normal contaminants concentration, accurate detection by bacteria using simple genetic circuit is very difficult. Consequently, CACCI is also established with systematic permeability reconstruction to increase its capability on molecules absoprtion.
Adhesion: According to our modeling, we need to adhere bacteria to a surface. Theoretically, when elastic deformation--caused by the movement of the bacteria containing cheZ parts--occurred on the film, interference will be observed.
In order to make bacteria firmly adhere to the film, two strategies for strong adhesion are taken into consideration, which including electrostatic interaction using polylysine(PLL) and covalent interaction by modifying bacterial transmembrane protein, which is based on the principle of clickable chemistry. Polylysine containing positive charge binds to gram-negative bacteria owing to their negative outer membrane. Consequently, strong electrostatic interaction between polylysine and bacterial membrane will take place theoretically. Moreover, covalent ligation will be established between the bacteria and the film to ensure stable adhesion. In order to achieve covalently adhesion, modification on bacterial transmembrane protein is necessary.
3.Package: In order to let everyone use the integrated improvement bacteria, CACCI in a more convenient way, we will provide CACCI in powder. We hope one day, iGEM could not only share parts, but also share device, a nanomachine with mature functional system.

Chemotaxis Modification Module

What is Chemotaxis?

Chemotaxis is a phenomenon of the movement of an organism, specific cells or bacteria, stimulated by chemical molecules. As for bacteria, naturally, chemoreceptors were expressed on the bacteria membrane, which enables the bacteria to 'sense' the existence of chemical molecule concentration in the surroundings. Chemotaxis consists of two situation, including positive chemotaxis and negative chemotaxis, which respectively demonstrates different behavior when the bacteria are exposed to surroundings with chemical molecules beneficial or harmful to their metabolism and reproduction.

The chemotaxis mechanism in E. coli can be illustrated as following:

When absence of chemical, there is no molecules bind to the chemoreceptors, cheW, an intracellular protein, is associated with cheA and cheA will autophosphorylate itself and capture phosphate group from ATP. Later, phosphate will be transferred to cheY and the phosphorylated cheY associates with the flagella motors making flagellum rotates clockwise. In general, bacteria tumble in the absence of chemical.
When presence of chemicals, cheA is free from cheW interaction, and cheZ will efficiently remove phosphate group from cheY. Those dephosphorylated cheY cannot bind with flagella motors, and consequently, flagella rotate counter-clock which can be observed as bacteria swim smoothly. In some special situations, cheR will methylate chemoreceptors to remain the disassociation between cheA and cheW. Then, bacteria will still swim but not tumble.

Genetically Engineered Chemotaxis Bacteria

Because of lack of natural chemoreceptors for antibiotics, we need to construct a genetic circuit specifically to sense antibiotics, and according to the mechanism of chemotaxis, we need to overexpress cheZ to improve its phosphorylation capability on cheY. The model is illustrated as following:

Integrative Modification on Bacterial Permeability

Our CACCI construction is systematically not only including chemotaxis modification, but containing some permeability improvement by inhibition of efflux system and overexpression of exogenous porin. Nonpolar residues face outward so as to interact with the nonpolar lipid membrane, whereas the polar residues face inwards into the center of the beta barrel to interact with the aqueous channel.

Porin: OprF from Pseudomonus aeruginosa

Porins are beta barrel proteins crossing a celluar membrane and act as a pore through which molecules are able to diffuse.

In E. coli different porins, for example OmpF and OmpC is expressed endogenously in different situations for different molecules. But these natural occurring porines are only permeable for molecules like sugars smaller than 600 Da. And more importantly, implement of micF, an antisense ncRNA may inhibits the material diffusion through OmpF. Therefore, it is necessary to import an exogenous porin with higher effiency of small molecular tranportation, such as OprF derived from Pseudomonas aeruginosa.

OprF represents one of the largest pore sizes on bacterial outer membranes, allowing diffusion of polysaccharides in a range of 2000 to 3000 Da. The overexpression of OprF in E. coli will drastically improve the antibiotic absorption capability, like antibiotic condensation occured in E. coli.

Viroporin: SCVE from SARS Virus

Viroporins are similar to porins in which they oligomerize within the membrane to form pores and this oligomerization often occurs between hydrophobic, transmembrane and alpha-helical domains of the proteins.

SCVE, SARS Caronavirus Envolope protein, is a kind of effective viroporin within 76 residues that is found normally pentamerized in bacteria. SCVE is permeable for many small molecules, which is originately designed for apoptosis of cells triggered by virus. Implementation of SCVE in ROSE may potentially improve the permatilibty of small molecules including antibiotics.

Inhibition of Efflux System using CRISPR

Efflux system is a kind of mechanism found in microbes responsible for many moving molecules, especially some toxic substances, microbiotics.

According to previous researches, efflux system contributes a lot for bacterial antibiotic resistance, or multidrug resistance(MDR). For example, in E. coli, AcrAB efflux system has a physiological role of pumping out many bile acids, fatty acids to lower their intracelluar toxicty. It would be an attemptation to go directly reverse to antibiotic resistance,which is to inhibit the function of efflux system. In order to completely inhibit the major efflux system of E. coli in genetic level, CRISPR would be an impressive solution for totally deletion of the function of efflux system.

Clutersted reularly interspacaed short palindromic repeats, abbreviated as CRISPR, are some fragements in prokayotic DNA with short repeats. Natually, CRISPR/Cas9 system is an important immune system that is able to resist the existence and reproduction of exogenous genetic materials, directly recognizing and then cut these genetic fragments, such as phages and plasmids, by CRISPR spacers, which is quite similar to RNA interference(RNAi) discovered in eukaryotic organisms. Cas9, which is CRISPR Associated Protein 9 in brief, is an RNA-guding DNA endonuclease enzyme associated with the CRISPR segments derived naturally from Streptococcus pyogenes and other bacteria. It has been shown that Cas9 plays an important role in memorizing, interrogating and ultimately cleaving these foreign DNA. Mechanism of cleavage is to unwind the foreign DNA and check whether there is a about 20 bp fragments which is complementary to guide RNA (gRNA, in brief). When the recognition result turns to yes, Cas9 will cleave the foreign DNA. Nowadays, with proper modification, CRIPSR/Cas9 has become a common gene editing tool, being able to cut the target gene with gRNA design.

When it comes to artificial design of CRISPR/Cas9 system, how to sucessfully achieve recruitment of Cas9 depended on the structure of gRNA. In general, gRNA consists of 2 main parts, the former part is about 20 bp RNA sequences, which is exactly complementary to target genes, and it will be more impressive if these complementary sequence is at the beginning of the transcrption sites of gene. And the latter part is a secondary structure part which finally forms as hairpin used to recruit Cas9. During engineered section, this structual part can directly import from iGEM parts and what we need to take care is the fragment complementary to efflux system gene.

CRISPR/Cas9 System for AcrB:

CRISPR/Cas9 System for EmrE:

Adhesion Module

What Material we Implement?-Theoretical Analysis

In order to produce slight deformation on the polymer film, we need to find a proper material to make it. According to our modeling, see more in Modeling-Film Candidate, pressure caused by adhesive bacteria is about 10^-4 Pa. Consequently, the wavelength of laser about 650nm is able to detect um-scale deformation. Plus considering cm squared film, we need the Young's Modulus of the film is less than 1GPa.

The Young's Modulus of polypropylene is about 1.5Gpa, so it is selected as our candidate polymer film.

How to Achieve Stable Adhesion?

Bacteria adhering to polymer film is one of the most important parts in CACCI. In order to make bacteria bind tightly, we provide two possible and promising solutions to figure it out, one of which is coating bacteria with polylysine(PLL) and the other is to chemically covalent bind between bacteria and polymer film.

Strategy I: Coated with Polylysine

Polylysine refers to lysine homopolymers, whose precursor amino acid lysine is composed of two amino groups, one at the α-carbon and one at the ε-carbon. Either can be the location of polymerization, resulting in α-polylysine or ε-polylysine.

α-Polylysine, a synthetic polymer, is composed of either L-lysine or D-lysine. "L" and "D" refer to the chirality at lysine's central carbon. This results in poly-L-lysine (PLL) and poly-D-lysine (PDL) respectively.

As a matter of fact, α-Polylysine is commonly used to coat tissue cultureware as an attachment factor which improves cell adherence. This phenomenon is based on the interaction between the positively charged polymer and negatively charged cells or proteins. This polycationic treatment on bacteria has already been proved with successful results when conducting microscopy experiment previously.

Figure 12: The adhesion of bacterial to polylysine

As for E. coli, a gram-negative bacterium, whose surface is formed by lipopolysaccharides(LPP), which is negatively charged at physiological pH. The polymer chain of polylysine is positively charged and remains in stochastic globule conformation at pH under 12.0. Thus, polylysine would readily absorb to film we designed and gives a smooth coverage in a form of monolayer, containing tightly packed globule with average lateral dimension of 20 nm. Polylysine is biocompatible and should not affect bacteria. To get more information on our adhesion assay, please refer to Results-Adhesion assay

Strategy II: Covalent Adhesion between bacterial membrane and film with proper modification.

To achieve covalently adhesion, we decided to implement the parts from 2014 iGEM TU_Eindhoven, and the whole system including these parts:

DBCO-PEG4-NHS Ester (Dibenocyclooctyne-Polyethylene glycol-N-Hydroxysuccinimide), which covalently binding to polymer film.

Figure 13: DBCO
pAzF(p-Azido-L-Phenylalanine), a rare amino acid that would be implemented in bacterial transmembrane protein biosynthesis.

Figure 14: pAzF
In order to introduce pAzF into bacterial transmembrane protein, we need to mutate one of transmembrane protein, which is called COMPX. COMPX is able to mutate with a special tRNA. The plasmid containing tRNA is as following:

Figure 15: tRNA plasmid

Covalent binding between DBCO and pAzF goes as following:

Figure 16:Schematic covalent interaction, this picture is extracted from guidebook of 2014 iGEM TU_Eindhoven

To see more about constitution of our adhesion system, please refer to Notebook-Protocol-Adhesion Preparation

Mutation of Transmembrane Protein OMPx in E. coli

OMPx is a integral outer membrane protein X from Escherichia coli, belonging to a family of highly conserved bacterial protein that promote bacterial adhesion to and entry into mammalian cells. Besides, these proteins as well play an important role in the resistance against attack by human complement system.

The modified COMPX structure is illustrated as below:

As planned, OMPx will be mutated at residue 16, from glycine to p-Azido-L-Phenylalanine, zooming in the mutated site in orange:

The Covalent Reaction Model

Consequently, the model of adhesion goes like this: modified bacteria containing special p-Azido-L-Phenylalanine transformed tRNA carry OMPx with mutated p-Azido-L-Phenylalanine site. The laminated side in pAzF will covalently bind to alkenyl in DBCO-PEG4-NHS ester, and consequently undergo chemical reactions.

Figure 19:The adhesion of bacterial to polymere

Package Your CACCI

This is a collaboration project between BIT. More information on our collaboration, see Collaboration. We really appreciate BIT for their energetic spirits and friendly.

Now, it's time to introduce why we need to package CACCI.

Because CACCI has been engineered in an integrated way, it contains both chemotaxis improvement and permeability modification. If you want to construct CACCI with strategy II, it requires one more plasmid in CACCI, which is obviously quite difficult for other iGEM team to extract plasmids from parts and transform them again. Consequently, we USTC decided to package our CACCI and share to everyone who need them.

Mechanism of packaging CACCI is quite simple, we firstly culture CACCI in LB liquid medium. (Attention: the LB liquid medium should contain glycerol which is used for cryopreservation. When bacteria grow in exponential phase. Get CACCI and culture them in skim milk containing sodium chloride. And we preserve those CACCI in refrigerator at minus 80 degree celcius for about 8 hours. Then put the CACCI into vacuum dryer, we would get CACCI in powder, ready for your recovery.

More details on how to package CACCI please visit: Protocol-Package your CACCI. See our results at Achievements-Package your CACCI

Bibliography

Comparative studies of bacteria with an atomic force microscopy operating in different modes. Ultramicroscopy 86 (2001) 121–128 A.V. Bolshakova, I.V. Yaminsky.
Measuring interfacial and adhesion forces between bacteria and mineral surfaces with biological force microscopy. PII S0016-7037(00)00430-0 Steven K. Lower, Michael F. hochella, JR.

Who is CACCI?
Chemotaxis Modification Module
Integrative Modification on Bacterial Permeability
Adhesion Module
Package Your CACCI
Bibliography

What's SPRING ?

Spring, which is named after its inventor, is CACCI's sidekick. He helps CACCI in three aspects, which are the optical aspect, the hardware aspect and the software aspect. Using Michelson Interferometer, the optical part, Spring transforms the shape change of CACCI's membrane into the change of interference fringe. Spring's hardware, based on Raspberry–Pi, allows spring to take photos or videos of the fringe. At last, the software analyses them and transmits the results to PC. CACCI depends on Spring.

Actually, SPRING is a measurement system which is designed to detect antibiotics in natural water bodies. As figure-1 shows, the system SPRING includes Optical part, Hardware part and Software part. Optical part is actually Michelson Interferometer, which can transform the shape change of a film into the change of interference fringe. Based on a Raspberry-Pi, hardware part can take photos or videos of interference fringe, then our software analyses them or transmits to your PC. Finally, we’ll see the results on the screen.

figure 1:The whole design of our electhic part and optical part

Why SPRING? Because we strongly call for limpid rives, just like spring flowing between mountains. Nowadays, contamination issue in river becomes more and more serious. Not only excessive antibiotic emission in China, lots of other toxic heavy metal ion, organic materials are found in river, which tremendously threaten public health and ecological balance. Consequently, we name our main body of device, SPRING, we hope with the assistance of our SPRING, we are able to share and enjoy those clean river, like those past days.

Michelson Interferometer

The Michelson interferometer is common configuration for optical interferometry and was invented by Albert Abraham Michelson. Using a beam-splitter, a light source is split into two arms. Each of those is reflected back toward the beam-splitter which then combines their amplitudes interferometrically. The resulting interference pattern that is not directed back toward the source is typically directed to some type of photoelectric detector or camera. Depending on the interferometer's particular application, the two paths may be of different lengths or include optical materials or components under test.
This is our design of optical part.

Hardware Module

Cooperating with the optical part, the electric circuit part makes use of a CCD camera to take pictures of interference fringe, and then our software in the Raspberry-Pi analyses them and display the results on the screen.

Figure 3 : The design of our hardware, looing from outside

This is the SPRING we built.

Electric Circuit

The Raspberry Pi is a low cost, credit-card sized computer that plugs into a computer monitor or TV, and uses a standard keyboard and mouse. It is a capable little device that enables people of all ages to explore computing, and to learn how to program in languages like Scratch and Python. It’s capable of doing everything you’d expect a desktop computer to do, from browsing the internet and playing high-definition video, to making spreadsheets, word-processing, and playing games. The module we used is Raspberry-PI 2B.

Figure 5: The real electric part we built

As the picture shows, the battery powers the little fan and the laser part. The little fan is necessary because of the need to lower the temperature of the board.

Figure 6: The electrical source and the transformer

The transformer transforms 12 volts to 5 volts, and the breadboard supplies power for the laser and the fan.

A breadboard is a construction base for prototyping of electronics. Originally it was literally a bread board, a polished piece of wood used for slicing bread. In the 1970s the solderless breadboard (AKA plugboard, a terminal array board) became available and nowadays the term "breadboard" is commonly used to refer to these. "Breadboard" is also a synonym for "prototype".

The screen, keyboard and the mouse are connected to the RaspberryPi via USB.

We use CCD camera to capture images.

A charge-coupled device (CCD) is a device for the movement of electrical charge, usually from within the device to an area where the charge can be manipulated, for example conversion into a digital value. This is achieved by "shifting" the signals between stages within the device one at a time. CCDs move charge between capacitive bins in the device, with the shift allowing for the transfer of charge between bins.

Optical Part: Michelson Interferometer

The Michelson interferometer is common configuration for optical interferometry and was invented by Albert Abraham Michelson. Using a beam-splitter, a light source is split into two arms. Each of those is reflected back toward the beam-splitter which then combines their amplitudes interferometrically. The resulting interference pattern that is not directed back toward the source is typically directed to some type of photoelectric detector or camera. Depending on the interferometer's particular application, the two paths may be of different lengths or include optical materials or components under test.

Figure 10: The real optical part we built

The following picture shows our details of optical part, which includes various splitters.

Beam splitter combination, consists of three mirrors, a 50% reflection - 50% transmission beam splitter (50%(R)/50%(T) in brief), a 80% reflection - 20% transmission beam splitter(80%(R)/20%(T) in brief) and a 10% reflection- 90% transmission beam splitter(10%(R)/90%(T) in brief). 1)50%(R)/50%(T) is in the front of picture. The reflection light will project to detecting film , while the transmission beam splitter will continue going to reflection mirror. 2) Then why do we need The 80%(R)/20%(T), along with 10%(R)/90%(T)? Actually these combination is really important to decrease the relatively high intensity reflected from reflector. Much higher intensity from reflector will not fit the formation condition of interference pattern. 80%(R)/20%(T)+10%(R)/90%(T), approximately allowing 18% light approach reflector, are combined together on the bottom of the picture.

Figure 12: The beam splitter used in the michelson interferometer

Mechanical part

We use a kind of plastic called Acrylic to build our mechanical part--the BOX. Acrylic is totally transparent while there is a piece of paper stuck on it. Our box was designed by ourselves and it's processed by company from the website TAOBAO.

And we use the electric drill to cut it. The head of the drill can be replaced by many part such as saws or polish-head.

Oh, all parts should be fixed with hot-melt adhesive (hot glue), in order to maintain stability. The glue is tacky when hot, and solidifies in a few seconds to one minute. Hot melt adhesives can also be applied by dipping or spraying.

Figure 15: One example of hot glue using

Software and Fringe analysis

Get your pictures! For example, you'll see this one.

Figure 16:An example of pictures you'll get using the CCD camera

And then analyze the picture. Wanna know how? Read on.

Figure 17: How to measure--the deformation of film

Methods

(1)

Take a series of photos at the same position in a short time.
Superpose these photos to sharp the edge of every object.
Choose two points in multi-image, the point must be on the black fringes.
Scanning these two fringes to find the shortest distance between them.
Calculate the radius and rank of every fringes.
Calculate the deformation of film.

(2)

Take a series of photos at the same position in a short time.
Superpose these photos to sharp the edge of every object.
Calculate the number of fringes on vertical direction(y axis).
Transform the number to deformation of the film.

More details on coding please refer to :(Add super link here)

This is our program, which was written in MATLAB. This program is used to simulate fringes pattern delivered by film I.

Code:

Demo

Figure 18:: Film II deformation simulation result

Using this code, we are able to capture some important parameters in Film I.

Code

Demo

Figure 19: Film I Fringes analysis running result

Cooperate with ROSE and CACCI

Along with ROSE and CACCI , SPRING will help detect antibiotic as well.

For details , you can look at this picture.

In order to measure the nano distance, we need to adhere bacteria to a surface, the film we used is processed cover slip.

Figure 22: Interference will be observed

During our experiment , when elastic deformation--caused by the movement of the bacteria containing cheZ parts--occurred on the film, interference will be observed.

Who is SPRING?
Michelson Interferometer
Hardware Module
Electric Circuit
Optical Part: Michelson Interferometer
Mechanical part
Software and Fringe analysis
Cooperate with ROSE and CACCIs

What is ROSE?

Reporter Operator with Sensing Engine, in brief-ROSE, is a systematically modified bacterial machine for antibiotic concentration determination. Because antibiotic substances contamination occurs in very low concentration, such as ~2000ng/L or 2ug/L that is much lower than normal contaminants concentration, accurate detection by bacteria using simple genetic circuit. Consequently, ROSE is established with both genetically amplified circuit and systematic permeability reconstruction.

Systematic Overview of ROSE

In general, ROSE as a community, containing two kinds of bacteria, among which Bacteria I specifically receives antibiotic concentration information from antibiotic substance sensing promoters, which were previously selected by Shimshon Belkin and Sahar Melamed in 2012. Then Bacteria I output information with LuxI, part of genetic expression regulator by quorum sensing, whose information is provided with positive amplification for Bacteria II. Bacteria II itself will automatically produce another part of regulator LuxR triggered by IPTG. Besides Bacteria II has a logically amplification circuit regulated by LuxI-LuxR complex. In this circuit containing two promoter regulating two protein. The former one is regulated by Lux complex and produces cI inhibition protein and the latter one will be inhibited by cI protein and EGFP will be expressed when lack of cI protein. Consequently, this genetic amplification circuit, compare with others, will not directly enhance the intensity of GFP expression but to increase the signal resolution of concentration variation.

The design intention on bacterial membrane permeability improvement is directly from part of mechanism of antibiotics resistance. Theoretically, bacteria won't receive antibiotic substance because of mutation of its transmembrane protein and efflux system. Thus, we are attempting to modify bacteria in reverse way, that is overexpression of transmembrane protein, or more specifically porin, and inhibition of efflux system production through CRISPR.

Antibiotic Substance Sensing Module

Previous research from Sahar Melamed and Shimshon Belkin has already selected dozens of promoters responding to antibiotic concentration specifically through the fluorescence signal.

Promoter Selection with Fluroescence Intensity, *2012 Shimshon Belkin*

For instance, micF has relatively specific signal response when treated with sulfamethoxazole(255.2), sulfadimethoxine(44.9) and colistin(36.9), soxS is able to sense tetracycline(26.5) and oxytetracycline(26.1) at relative high intense. Besides, recA will effectively detect the existence of nalidixic acid and etc. These maximal induction of each promoter strain is tested in the course of 10h of exposure.

The mechanism of these promoters are different from each other. However, basically, mechanisms consist of: - micF, an antisense ncRNA targeting ompF RNA, main endogenous porin of Escherichia coli, participating in post-transcriptionally regulation of this outer membrane protein F(ompF). micF is also regarded as one of the promoter of soxRS-regulated gene.

micF: antisense ncRNA with ompF mRNA 5' UTR,*2011 JMB*

SoxS, a transcriptional activator of oxidative stress genes in E. coli. SoxS in vitro binds the promoter of soxRS-regulated genes such as micF, sodA and recruits RNA polymerase to the promoters.

In accordance with relative effective response of antibiotics, we are determined to implement micF to sense the existence of sulfamethoxazole and sulfadimethoxine in water sample,

and SoxS to detect the concentration of tetracycline or oxytetracycline.

In the circuit, micF and SoxS won't directly reporter fluorescence signal, instead, information will deliver through quorum sensing for positive amplification. In antibiotic substance sensing module, LuxI will be expressed directly for Bacteria II processing.

Quorum Sensing Module

Using community with different compartments would further improve the signal amplification, as known, quorum sensing is a system for stimulation(some signaling molecules called autoinducers) and response(receptor that will transcript certain genes) that is related to the bacterial concentration in the system. Many gene expression is naturally regulated by quorum sensing, which is also treated as social interaction in bacterial community.

In some gram-negative bacteria, such as Vibro fischeri, biosynthesis of autoinducers is used for yield of bioluminescence through luciferase. The signaling molecule used by V. fischeri is an acylated homoserine lactone(AHL),called N-(3-oxohexanol)-homoserine lactone, which can be engineered into E. coli. The signaling molecule will be produced in the cytoplasm using LuxI synthase enzyme and then secreted through the cell membrane to the extracellular surroundings. When N-(3-oxohexanol)-homoserine lactone begins diffusing back into cells or other bacteria, LuxR will recognize the existence of N-(3-oxohexanol)-homoserine lactone with a threshold concentration, about 10ug/mL. Finally, target gene such as LuxlCDABE will be activated with LuxR and N-(3-oxohexanol)-homoserine lactone complex.

It has been proved that LuxR has evolutionary conservation in many gram-negative bacteria, which enables E. coli to receive AHL from other bacteria. Thus we engineer Bacteria I to release quorum sensing molecules to Bacteria II for exponentially amplified results.

To achieve antibiotic concentration corresponding to AHL production, AHL produced by Bacteria I will be regulated by antibiotic substance sensing promoters. And in Bacteria II, LuxR will be directly expressed regulated by lac operon, triggered by IPTG. With treatment of IPTG, LuxR will be constitutively expressed to ensure LuxR-AHL complex will initiate the expression of required genes.

Second Level Amplification Module

In addition of signal amplification through quorum sensing, an artificial genetically engineered cascade magnification circuit is also implemented in ROSE construction.

In this cascade signal circuit, LuxR-AHL complex trigger the activation of promoter Lux, then express cI derived from λ phage. Since cI is a highly efficient repressor that is a low concentration of cI will completely repress the efficiency of λ promoter. As a result, EGFP will be drastically and negatively regulated when treated with antibiotic substances.

It is notable that this second level amplification module won't directly expand the signal intensity, in another way, it will indirectly enlarge the resolution of different antibiotic concentration gradients. Our theoretical modeling also illustrate a higher resolution implementing this amplification circuit.

Integrative Modification on Bacterial Permeability

Our ROSE construction is systematically not only include quorum sensing and logic amplification, but containing some permeability improvement by inhibition of efflux system and overexpression of exogenous porin. Nonpolar residues face outward so as to interact with the nonpolar lipid membrane, whereas the polar residues face inwards into the center of the beta barrel to interact with the aqueous channel.

Porin: OprF from Pseudomonus aeruginosa Porins are beta barrel proteins crossing a celluar membrane and act as a pore through which molecules are able to diffuse.

In E. coli different porins, for example OmpF and OmpC is expressed endogenously in different situations for different molecules. But these natural occurring porines are only permeable for molecules like sugars smaller than 600 Da. And more importantly, implement of micF, an antisense ncRNA may inhibit the material diffusion through OmpF. Therefore, it is necessary to import an exogenous porin with higher effiency of small molecular tranportation, such as OprF derived from Pseudomonas aeruginosa.

OprF represents one of the largest pore sizes on bacterial outer membranes, allowing diffusion of polysaccharides in a range of 2000 to 3000 Da. The overexpression of OprF in E. coli will drastically improve the antibiotic absorption capability, like antibiotic condensation occured in E. coli.

Viroporin: SCVE from SARS Virus Viroporins are similar to porins in that they oligomerize within the membrane to form pores and this oligomerization often occurs between hydrophobic, transmembrane and alpha-helical domains of the proteins.

SCVE, SARS Caronavirus Envolope protein, is a kind of effective viroporin within 76 residues that is found normally pentamerized in bacteria. SCVE is permeable for many small molecules, which is originately designed for apoptosis of cells triggered by virus. Implementation of SCVE in ROSE may potentially improve the permatilibty of small molecules including antibiotics.

Inhibition of Efflux System using CRISPR Efflux system is a kind of mechanism found in microbes responsible for many moving molecules, especially some toxic substances, microbiotics.

According to previous research, efflux system contributes a lot for bacterial antibiotic resistance, or multidrug resistance(MDR). For example, in E. coli, AcrAB efflux system has a physiological role of pumping out many bile acids, fatty acids to lower their intracelluar toxicty. It would be an attemptation to go directly reverse to antibiotic resistance, that is to inhibit the function of efflux system. In order to completely inhibit the major efflux system of E. coli in genetic level, CRISPR would be an impressive solution for totally deletion of the function of efflux system.

Clutersted reularly interspacaed short palindromic repeats, abbreviated as CRISPR, are some fragements in prokayotic DNA with short repeats. Natually, CRISPR/Cas system is an important immune system that is able to resist the existence and reproduction of exogenous genetic materials, directly recognizing and then cut these genetic fragments, such as phages and plasmids, by CRISPR spacers, which is quite similar to RNA interference(RNAi) discovered in eukaryotic organisms. Cas9, which is CRISPR Associated Protein 9 in brief, is an RNA-guding DNA endonuclease enzyme associated with the CRISPR segments derived naturally from * Streptococcus pyogenes* and other bacteria. It has been showed that Cas9 plays an important role in memorizing, interrogating and ultimately cleaving these foreign DNA. Mechanism of cleavage is to unwind the foreign DNA and check whether there is a about 20 bp fragments that is complementary to guide RNA (gRNA, in brief). When the recognition result turns to yes, Cas9 will cleave the foreign DNA. Nowadays, with proper modification, CRISPR/Cas9 has become a common gene editing tool, being able to cut the target gene with gRNA design.

When it comes to artificial design of CRISPR/Cas9 system, how to sucessfully achieve recruitment of Cas9 depends on the structure of gRNA. In general, gRNA consists of 2 main parts, the former part is about 20 bp RNA sequences, which is exactly complementary to target genes, and it will be more impressive if these complementary sequence is at the beginning of the transcrption sites of gene. And the latter part is a secondary structure part which finally forms as hairpin used to recruit Cas9. During engineered section, this structual part can directly import from iGEM parts and what we need to take care is the fragment complementary to efflux system gene.

CRISPR/Cas9 System for AcrB:

CRISPR/Cas9 System for EmrE:

Theoretical Advantage

Bibliography

1.A bacterial reporter panel for the detection and classification of antibiotic substances Microbial Biotechnology (2012) 5(4), 536-548Sahar Melamed, Shimshon Belkin

2.Signal-Amplifying Genetic Circuit Enables In Vivo Observation of WeakPromoter Activation in the Rhl Quorum Sensing SystemWiley InterScience 2005DOI: 10.1002/bit.20371

3.MicF: An Antisense RNA Gene Involved in Response of Escherichia coli to Global Stress Factors 2001 JMB doi:10.1006/jmbi.2001.5029

What is ROSE?
Systematic Overview
of ROSE
Antibiotic Substance
Sensing Module
Quorum Sensing Module
Second Level
Amplification Module
Integrative Modification
on Bacterial Permeability
Theoretical Advantage
Bibliography

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-                                <img src="./Overview.png" style="width:250px;">
-                             </div>
+                             <h4 id="ndm-nanomachine-detecting-microbiotics" class="scrollspy">NDM: Nanomachine Detecting
-                            <div class="divider"></div>
+                                Microbiotics</h4>
-                            <h3><b id="OverviewNDM" class="scrollspy">NDM:</b><br /><b>Nanomachine Detecting Microbiotics</b></h3>
+                             <p>Abusing antibiotics has caused severe antibiotics contamination and resistance issues
-                             <p>
+                                worldwide. Therefore, we USTC develop a device NDM with an optical interference path,
-                                Abusing antibiotics has caused severe antibiotics contamination and resistance issues worldwide. Therefore, we USTC develop a device NDM with OD detector and optical interference path, recognition program based on RasberryPi and Arduino to detect antibiotics in natural water bodies.
+                                recognition program based on RasberryPi and Arduino to detect antibiotics in natural
-                                 <br />
+                                water bodies. In NDM, there is a system for measurement: CACCI which contains chemotaxis
-                                 <br />
+                                 modified bacterial adhered to a polymer membrane. Deformation caused by CACCI motility
-                                 In NDM, there are two bacterial systems for measurement:
+                                can be recognized with optical interference and the interference pattern recognition
-                                 <br />
+                                program inside NDM will analyze the deformation, thus antibiotic situation in sample can
-                                 <br />
+                                be accurately told. </p>
-                                 <b id="OverviewROSE" class="scrollspy">ROSE</b>, an engineered bacterial reporter system integrated with permeability modification, logic amplification circuit and quorum sensing, is able to adjust EGFP expression level, and NDM reads fluorescence intensity from ROSE to send results in high resolution.
+                                 <div class="divider"></div>
-                                 <br />
+                            <h4 id="what-is-antibiotics-" class="scrollspy">What is Antibiotics?</h4>
-                                 <br />
-                                 <b id="OverviewCACCI" class="scrollspy">CACCI</b>, chemotaxis and transmembrane protein-modified bacteria, are chemically covalently adhered to a polymer membrane. Deformation caused by CACCI motility can be recognized with optical interference and the interference pattern recognition program inside NDM will analyze the deformation, thus antibiotic situation in sample can be accurately told.
+                            <p>Antibiotics, or microbiotics, in narrow sense, are a various type of artificially
-                                 <br /> Hope with our project, antibiotics issues can be solved in an accelerated way.
+                                 constructed or naturally existing chemicals containing the capability of killing
-                            </p>
+                                bacteria or inhibiting their growth, which are implemented widespread around the
+                                world. Patients who got bacterial or protozoan infection from surgical wound like war
+                                field, empyrosis and previously regarding severe diseases such as general cold,
+                                tuberculosis(TB), sexual transmitted diseases(STD), dysentery, anthrax, even cancer and
+                                so forth are able to cure treating with antibiotic substances. Since the discovery of
+                                penicillin by British biologists Alexander Flaming in 1929, the antibiotic family has
+                                been enlarged a lot. With properly administration of antibiotics, millions of people
+                                were saved in evidently alleviated symptom. There was a time when antibiotics, along
+                                with oil source for automobile, was indispensable for human beings.</p>
+                            <p>Antibiotic substances containing several mechanism, rendering them to kill and inhibit
+                                bacterial growth in their own ways. Those mechanisms including:</p>
+                            <ul>
+                                 <li>Inhibit the synthesis of cell wall.</li>
+                                <li>Influence the function of cell membrane.</li>
+                                <li>Inhibit the biosynthesis of nucleic acid.</li>
+                                <li>Inhibit the biosynthesis of proteins.</li>
+                            </ul>
+                            <br>Classes categorization based on chemical structure
+                                including:</p>
+                            <ul>
+                                 <li>Beta-lactam</li>
+                                <li>Aminoglycoside</li>
+                                <li>Amide alcohol</li>
+                                <li>Macrolide</li>
+                                <li>Polypeptide</li>
+                                <li>Tetracycline</li>
+                                <li>Sulfonamides</li>
+                                <li>Quinolones</li>
+                                <li>Polymyxin</li>
+                                <li>Trimethoprim</li>
+                                <li>Nitrofuran etc.</li>
+                            </ul>
+                            <p>Ordered formal course of treatment, these antibiotic would effectively treat<br>gonorrhea,
+                                MRSA and pseudomonal infections respectively.</p>
+                            <p>However, treating with microbiotics may be associated with a rang of side effects, from
+                                 some mild including gastrointestinal upset, diarrhea to severe like hearing loss, kidney
+                                damage. Some speculation from scientists postulated that exposition of high
+                                concentration utilization of antibiotics alter the host microbiota, especially the
+                                family of the probiotics, and this has been associated with chronic diseases.</p>
+                            <p>The production of antibiotics in pharmaceutical companies explosively expanded these
+                                years. Artificial synthesis of sulfonamides, quinolones and oxazolidinones enrich the
+                                categories of antibiotics.</p>
+                                 <div class="divider"></div>
+                            <h4 id="antimicrobial-resistance-and-abuses" class="scrollspy">Antimicrobial Resistance and
+                                Abuses</h4>
+                            <p>As the matter of fact, microbiotics, the great saver of our life, is now putting us in
+                                potentially tremendous disaster both environmentally and physiologically. </p>
+                            <p>Emergence of antimicrobial resistance is theoretically predictable by evolutionary
+                                potential. The antibiotic treatment may select for bacterial strains physiologically
+                                improved of their cell bodies or genetically enhanced capacity of mutagenesis and gene
+                                horizontal transfer like conjugation.</p>
+                            <p>Because of such horizontal genetic exchange, nowadays, efficacy loss may attribute to the
+                                emergence of many antimicrobrial-resistent strains. For instance, tuberculosis(TB) in
+                                China becomes more difficult with administration with previous antibiotic substances
+                                owing to elevating level of antimicrobrial resistance, which raised great challenge on
+                                Chinese public health. Besides, the discovery of NDM-1(New Delhi Metallo-beta-lactamase)
+                                in <em>Klebsiella pneumoniae</em> from a Swedish patient of Indian origin in 2008 once
+                                 may contribute to severe disaster on that broad of beta-lactam antibiotics.</p>
+                            <p>Though, antimicrobial resistance can be transferred through evolutionary theory, main
+                                 contribution of increasing antibiotic resistance comes from individuals' inappropriate
+                                antibiotics treatment, overuse of antibiotics without prescription from professional
+                                doctor, instead from self-prescription.</p>
+                            <p>Demonstrably, attribution of antibiotic resistance not only from clinically, but from
+                                 agriculture and animal husbandry. In China, the overuse of antibiotics for animal is the
+                                same serious as the clinical therapy. In farms or hospitals, solution containing
+                                antibiotics directly emits to river, which potentially strongly influence the
+                                ecological equilibrium of bactierial colonies and sidelong threaten public health.</p>
+                                 <div class="divider"></div>
+                            <h4 id="antibiotics-production-and-contamination-in-china" class="scrollspy">Antibiotics
+                                Production and
+                                Contamination in China</h4>
+                            <p>Because individuals neglecting antibiotic emission, rivers in China now encounter
+                                extremely antibiotic contamination.</p>
+                            <p>According to the latest research from <em>Chinese Academy of Science</em> published on
+                                ACS Environmental Science and Technology, China used 162,000 tons of antibiotic
+                                substances for human(48%) and animal(52%) in 2013, which is tenfold usage of USA and
+                                antibiotic substances in high concentration can be detected from many river basins that
+                                may potentially harmful to public health and environment. In their professional
+                                evaluation, antibiotics can be detected in all rivers in China in different degree. </p>
+                            <p>Among these rivers, the Pearl River Basin in South China and Hai River Basin in North
+                                China received the highest antibiotics emission. More than 36 common antibiotic
+                                substances were discovered in these rivers, one of them, for example, in the Pearl River
+                                Basin, amoxicillin was found at 3384 ng/L and fluprofen at 2867 ng/L. Though lack of
+                                standard ranking on antibiotic contamination, according to environmental scientists
+                                from CAS, antibiotic substances concentration more than 1000 ng/L is relatively
+                                extremely high, based on Europe Union Standard.</p>
+                            <p>Now, many technology including ELISA, Delvotest, HPLC-MS/MS, CE and Quantum Dot are able
+                                to detect antibiotic concentration in river sample, however much of their have much
+                                deficiencies, including:</p>
+                            <ul>
+                                <li>Not portable, many big and automatic equipment are indispensable for concentration
+                                    detection.
+                                </li>
+                                <li>Complicated Routine, which requires trained personnel to conduct experiment. However,
+                                    facing that large scale contamination, complicated routine and trained personnel
+                                    cannot be satisfied at all districts, so do it in many developing and undeveloped
+                                    countries and districts.
+                                 </li>
+                                <li>Recent technology is still not sensitive enough and sometimes time consuming.</li>
+                            </ul>
+                            <p>Hope with our project, antibiotics issues can be solved in an accelerated way.</p>
                          </div>
                      </div>
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                              <ul class="section table-of-contents">
                                  <li>
-                                     <a href="#OverviewNDM">Overview:NDM</a>
+                                     <a href="#ndm-nanomachine-detecting-microbiotics">NDM: Nanomachine Detecting
+                                        Microbiotics</a>
+                                </li>
+                                <li>
+                                    <a href="#what-is-antibiotics-">What is Antibiotics?</a>
                                  </li>
                                  <li>
-                                     <a href="#OverviewROSE">Overview:ROSE</a>
+                                     <a href="#antimicrobial-resistance-and-abuses">Antimicrobial Resistance and
+                                        Abuses</a>
                                  </li>
                                  <li>
-                                     <a href="#OverviewCACCI">Overview:CACCI</a>
+                                     <a href="#antibiotics-production-and-contamination-in-china">Antibiotics Production
+                                        and
+                                        Contamination in China</a>
                                  </li>
                              </ul>
@@ Line 85: / Line 214: @@
              </div>
          </div>
-         <div id="Background" class="row">
+         <div id="CACCI" class="row">
              <div class="card hoverable">
                  <div class="row">
                      <div class="col s12 m9">
                          <div class="card-content">
-                             <h3 id="what-is-antibiotics-" class="scrollspy">What is Antibiotics?</h3>
+                             <h4 id="who-is-cacci" class="scrollspy">Who is CACCI?</h4>
-                             <p>Antibiotics, or microbiotics, in narrow sense, are a various type of artificially constructed or naturally existing chemicals containing the capability of killing bactiera or inhibiting their growth, which are implemented widespreadly around the world. Patients who got bactieral or protozoan infection from surgical wound like war field, empyrosis and previously regarding severe diseases such as general cold, tuberculosis(TB), sexual transmitted diseases(STD), dysentery, anthrax, even cancer and so forth are able to cure treating with antibiotic substances. Since the discovery of penicillin by British biologists Alexander Flaming in 1929, the antibiotic family has been enlarged a lot. With properly administration of antibiotics, millions of people were saved in evidently alleviated symptom. There was a time when antibiotics, along with oil source for automobile, was indispensable for human beings.</p>
-                            <br />
+                             <p><strong><em>CACCI</em></strong>, called the same as Kathy, is a kind of constructed
-                             <p>Antibiotic substances containing several mechanism, rendering them to kill and inhibit bactieral growth in their own ways. Those mechanisms including:</p>
+                                bacteria for <strong><em>Characterization of Antibiotic Concentration based on
-                             <ul class="collection">
+                                    Chemotaxis and Interference.</em></strong></p>
-                                 <li class="collection-item">inhibit the synthesis of cell wall.</li>
-                                <li class="collection-item">influence the function of cell membrane.</li>
+                            <p>Along with ROSE, CACCI embedded in SPRING will be used to detect antibiotic. Although,
-                                <li class="collection-item">inhibit the biosynthesis of nucleic acid.</li>
+                                the construction of ROSE will specifically tell the concentration of different
-                                <li class="collection-item">inhibit the biosynthesis of proteins.</li>
+                                antibiotics in the river samples, its feature is also deficiency: that is such strong
-                            </ul>
+                                specificity may hurt extensive measurement. Besides, using traditional genetically
-                             <p><img src="https://upload.wikimedia.org/wikipedia/commons/0/08/Antibiotics_Mechanisms_of_action.png" alt="From TheMedSchool">
+                                modified circuits to express report is still time-consuming, comparing with general
-                                <br>Classes catogorization based on chemical structure including:</p>
+                                physical or chemical measurement approaches.</p>
-                             <ul class="collection">
-                                <li class="collection-item">Beta-lactam</li>
+                             <p>Consequently, we originally established CACCI based on chemotaxis and interference to
-                                <li class="collection-item">Aminoglycoside</li>
+                                make a larger level of antibiotic molecule detection, even a detection for other
-                                <li class="collection-item">Amide alcohol</li>
+                                chemical molecules. So, the mechanism and construction of CACCI can be divided into the
-                                <li class="collection-item">Macrolide</li>
+                                following schedules:</p>
-                                <li class="collection-item">Polypetide</li>
+                             <ol>
-                                <li class="collection-item">Tetracycline</li>
+                                 <li><strong>Chemotaxis Modification</strong>: to increase the bacterial mobility, modification based on <em>E. coli</em> chemotaxis related to the parts cheZ will be introduced into bacteria.</li>
-                                <li class="collection-item">Sulfonamides</li>
+                            <li><strong>Permeability Improvement</strong>: Because antibiotic substances contamination occurs in very low concentration, such as ~2000ng/L or 2ug/L that is much lower than normal contaminants concentration, accurate detection by bacteria using simple genetic circuit is very difficult. Consequently, CACCI is also established with systematic permeability reconstruction to increase its capability on molecules absoprtion.</li>
-                                <li class="collection-item">Quinolones</li>
+                            <li><strong>Adhesion</strong>: According to our modeling, we need to adhere bacteria to a surface. Theoretically, when elastic deformation--caused by the movement of the bacteria containing cheZ parts--occurred on the film, interference will be observed.
-                                <li class="collection-item">Polymyxin</li>
+                                <br>In order to make bacteria firmly adhere to the film, two strategies for strong adhesion are taken into consideration, which including <strong>electrostatic interaction using polylysine(PLL)</strong> and <strong>covalent interaction by modifying bacterial transmembrane protein</strong>, which is based on the principle of clickable chemistry. Polylysine containing positive charge binds to gram-negative bacteria owing to their negative outer membrane. Consequently, strong electrostatic interaction between polylysine and bacterial membrane will take place theoretically. Moreover, covalent ligation will be established between the bacteria and the film to ensure stable adhesion. In order to achieve covalently adhesion, modification on bacterial transmembrane protein is necessary.
-                                <li class="collection-item">Trimethprim</li>
+                                <br>3.<strong>Package</strong>: In order to let everyone use the integrated improvement bacteria, CACCI in a more convenient way, we will provide CACCI in powder. We hope one day, iGEM could not only share parts, but also share device, a nanomachine with mature functional system.</li>
-                                <li class="collection-item">Nitrofuran etc.</li>
+                        </ol>
-                            </ul>
-                            <p><img src="http://gardeningblogger.info/image/55572f8e4a771.jpg" alt=""></p>
+                            <div class="divider"></div>
-                             <p>Ordered formal course of treatment, these antibiotic would effectly treat
+                            <h4 id="chemotaxis-modification-module" class="scrollspy">Chemotaxis Modification
-                                <br>gonorrhea, MRSA and pseudomonal infections respectively.</p>
+                                Module</h4>
-                            <br/>
-                            <p>However, treating with microbiotics may be associated with a rang of side effects, from some mild including gastrointestinal upset, diarrhea to severe like hearing loss, kidney damage. Some speculation from scientists postulated that exposition of high concentration utilization of antibiotics alter the host microbiota, especially the familiy of the probiotics, and this has been associated with chronic diseases.</p>
+                            <p><strong>What is Chemotaxis?</strong></p>
-                            <br />
+                            <p>Chemotaxis is a phenomenon of the movement of an organism, specific cells or bacteria, stimulated by chemical molecules. As for bacteria, naturally, chemoreceptors were expressed on the bacteria membrane, which enables the bacteria to 'sense' the existence of chemical molecule concentration in the surroundings. Chemotaxis consists of two situation, including positive chemotaxis and negative chemotaxis, which respectively demonstrates different behavior when the bacteria are exposed to surroundings with chemical molecules beneficial or harmful to their metabolism and reproduction.</p>
-                            <p>The prodcution of antibiotics in pharmceutical companies explosively expanded these years. Articifical synthesis of sulfonamides, quinolones and oxazolidinones enrich the categories of antibiotics.</p>
+                        <p>The chemotaxis mechanism in <em>E. coli</em> can be illustrated as following:</p>
-                            <br />
-                            <p class="divider"></p>
+                        <ul>
-                            <br />
+                             <li>
-                            <h3 id="antimicrobial-resistance-and-abuses" class="scrollspy">Antimicrobial Resistance and Abuses</h3>
+                                <p>When absence of chemical, there is no molecules bind to the chemoreceptors, cheW, an intracellular protein, is associated with cheA and cheA will autophosphorylate itself and capture phosphate group from ATP. Later, phosphate will be transferred to cheY and the phosphorylated cheY associates with the flagella motors making flagellum rotates clockwise. In general, bacteria tumble in the absence of chemical.</p>
-                            <p>As the matter of fact, microbiotics, the great saver of our life, is now putting us in potentially tremendous disaster both environmentally and physiologically. </p>
+                            </li>
-                            <br />
+                            <li>
-                             <p>Emergence of antimicrobial resistance is theoratically predictable by evolutionary potential. The antibiotic treatment may select for bacterial strains physiologically improved of their cell bodies or genetically enhanced capacity of mutagenesis and gene horizontal transfer like conjugation.</p>
+                                <p>When presence of chemicals, cheA is free from cheW interaction, and cheZ will efficiently remove phosphate group from cheY. Those dephosphorylated cheY cannot bind with flagella motors, and consequently, flagella rotate counter-clock which can be observed as bacteria swim smoothly. In some special situations, cheR will methylate chemoreceptors to remain the disassociation between cheA and cheW. Then, bacteria will still swim but not tumble.</p>
-                            <br />
+                            </li>
-                            <p>Because of such horizontal genetic exchange, nowadays, efficacy loss may attribute to the emergence of many antimicrobrial-resistent strains. For instance, tuberculosis(TB) in China becomes more difficult with administration with previous antibiotic substances owing to elevating level of antimicribrial resistence, which raised great challenge on Chinese public health. Besides, the discovery of NDM-1(New Delhi Metallo-beta-lactamase) in <em>Klebsiella pneumoniae</em> from a Swedish patient of Indian origin in 2008 once may contribute to severe disaster on that broad of beta-lactan antibiotics.</p>
+                        </ul>
-                            <br />
+                        <p><strong>Genetically Engineered Chemotaxis Bacteria</strong></p>
-                            <p>Though, antimicrobial resistance can be transferred through evolutionary theory, main contribution of increasing antibiotic resistance comes from individuals' inappropriate antibiotics treatment, overuse of antibiotics without prescription from professional doctor, instead from self-prescription.</p>
+                        <p>Because of lack of natural chemoreceptors for antibiotics, we need to construct a genetic circuit specifically to sense antibiotics, and according to the mechanism of chemotaxis, we need to overexpress cheZ to improve its phosphorylation capability on cheY. The model is illustrated as following:</p>
-                            <p><img src="http://chuantu.biz/t2/11/1438322045x-954498856.jpg" alt="图片名称"></p>
-                            <p>Demonstrably, attribution of antibiotic resistance not only from clinically, but from agriculture and animal husbandry. In China, the overuse of antibiotics for animal is the same serious as the clinical therapy. In farms or hospitals, solution containing antibiotics directly emittes to river, which potentially strongly influence the ecological equilibrium of bactierial colonies and sidelong threaten public health.</p>
+                             <div class="divider"></div>
-                            <br />
+                            <h4 id="integrative-modification-on-bacterial-permeability" class="scrollspy">Integrative Modification on Bacterial Permeability</h4>
-                             <p class="divider"></p>
+                        <p>Our CACCI construction is systematically not only including chemotaxis modification, but containing some permeability improvement by inhibition of efflux system and overexpression of exogenous porin. Nonpolar residues face outward so as to interact with the nonpolar lipid membrane, whereas the polar residues face inwards into the center of the beta barrel to interact with the aqueous channel.</p>
-                             <br />
+                        <p><strong>Porin: OprF from <em>Pseudomonus aeruginosa</em></strong></p>
-                             <h3 id="antibiotics-production-and-contamination-in-china" class="scrollspy">Antibiotics Production and Contamination in China</h3>
+                        <p>Porins are beta barrel proteins crossing a celluar membrane and act as a pore through which molecules are able to diffuse. </p>
-                             <p>Because individuals neglecting antibiotic emission, rivers in China now encounter extremely antibiotic contamination.</p>
+                        <p>In <em>E. coli</em> different porins, for example OmpF and OmpC is expressed endogenously in different situations for different molecules. But these natural occurring porines are only permeable for molecules like sugars smaller than 600 Da. And more importantly, implement of micF, an antisense ncRNA may inhibits the material diffusion through OmpF. Therefore, it is necessary to import an exogenous porin with higher effiency of small molecular tranportation, such as OprF derived from <em>Pseudomonas aeruginosa</em>.</p>
-                            <p><img src="image/1438323729x-1566638187.png" alt="图片名称"></p>
-                             <p>According to the latest research from <em>Chinese Academy of Science</em> published on ACS Environmental Science and Technology, China used 162,000 tons of antibiotic substances for human(48%) and animal(52%) in 2013, which is tenfold usage of USA and antibiotic substances in high concentration can be detected from many river basins that may potentially harmful to public health and environment. In their professional evaluation, antibiotics can be detected in all rivers in China in different degree. </p>
+                        <p>OprF represents one of the largest pore sizes on bacterial outer membranes, allowing diffusion of polysaccharides in a range of 2000 to 3000 Da. The overexpression of OprF in <em>E. coli</em> will drastically improve the antibiotic absorption capability, like antibiotic condensation occured in <em>E. coli</em>.</p>
-                            <p><img src="image/1438322929x-1566638187.png" alt="图片名称"></p>
+                        <p><strong>Viroporin: SCVE from SARS Virus</strong></p>
-                            <p>Among these rivers, the Pearl River Basin in South China and Hai River Basin in North China received the highest antibiotics emission. More than 36 common antibiotic substances were discovered in these rivers, one of them, for example, in the Pearl River Basin, amoxicillin was found at 3384 ng/L and fluprofen at 2867 ng/L. Though lack of standard ranking on antibiotic contamination, according to environmental scienctists from CAS, antibiotic substances concentration more than 1000 ng/L is relatively extremely high, based on Europe Union Standard.</p>
+                        <p>Viroporins are similar to porins in which they oligomerize within the membrane to form pores and this oligomerization often occurs between hydrophobic, transmembrane and alpha-helical domains of the proteins.</p>
-                            <br />
-                            <p>Now, many technology including ELISA, Delvotest, HPLC-MS/MS, CE and Quantum Dot are able to detect antibiotic concentration in river sample, however much of their have much dificiencies, including:</p>
+                        <p>SCVE, SARS Caronavirus Envolope protein, is a kind of effective viroporin within 76 residues that is found normally pentamerized in bacteria. SCVE is permeable for many small molecules, which is originately designed for apoptosis of cells triggered by virus. Implementation of SCVE in ROSE may potentially improve the permatilibty of small molecules including antibiotics.</p>
-                            <ul class="collection">
+                        <p><strong>Inhibition of Efflux System using CRISPR</strong></p>
-                                <li class="collection-item">Not portable, many big and automatic equipments are indispensable for concentration detection.</li>
+                        <p>Efflux system is a kind of mechanism found in microbes responsible for many moving molecules, especially some toxic substances, microbiotics.</p>
-                                <li class="collection-item">Complicated Routine, which requires traned personnel to conduct experiment. However, facing that large scale contamination, complicated routine and trained personnel cannot be satisfied at all districts, so do it in many developing and undeveloped countries and districts.</li>
-                                <li class="collection-item">Recent technology is still not sensitive enough and sometimes time consuming.</li>
+                            <figure>
-                            </ul>
+                              <img src="https://static.igem.org/mediawiki/2015/d/d2/Strucuture_of_EmrE%2C_3B61_in_PDB.png">
-                            <p>Consequently, this year we iGEM constructed NDM, which is abbreviated from Nanomachine detecting microbiotics. Using NDM based on mechanism of GFP reporter, chemotaxis and interference, a portable and accurate device is established to encounter the issues today.</p>
+                              <figcaption>
-                            <p><a onclick="$(document).scrollTop(0)">Top</a></p>
+                              Strucuture of EmrE, 3B61 in PDB
+                              </figcaption>
+                             </figure>
+                        <p>According to previous researches, efflux system contributes a lot for bacterial antibiotic resistance, or multidrug resistance(MDR). For example, in <em>E. coli</em>, AcrAB efflux system has a physiological role of pumping out many bile acids, fatty acids to lower their intracelluar toxicty. It would be an attemptation to go directly reverse to antibiotic resistance,which is to inhibit the function of efflux system. In order to completely inhibit the major efflux system of <em>E. coli</em> in genetic level, CRISPR would be an impressive solution for totally deletion of the function of efflux system.</p>
+                        <p>Clutersted reularly interspacaed short palindromic repeats, abbreviated as <em>CRISPR</em>, are some fragements in prokayotic DNA with short repeats. Natually, CRISPR/Cas9 system is an important immune system that is able to resist the existence and reproduction of exogenous genetic materials, directly recognizing and then cut these genetic fragments, such as phages and plasmids, by CRISPR spacers, which is quite similar to RNA interference(RNAi) discovered in eukaryotic organisms. Cas9, which is CRISPR Associated Protein 9 in brief, is an RNA-guding DNA endonuclease enzyme associated with the CRISPR segments derived naturally from <em> Streptococcus pyogenes</em> and other bacteria. It has been shown that Cas9 plays an important role in memorizing, interrogating and ultimately cleaving these foreign DNA. Mechanism of cleavage is to unwind the foreign DNA and check whether there is a about 20 bp fragments which is complementary to guide RNA (gRNA, in brief). When the recognition result turns to yes, Cas9 will cleave the foreign DNA. Nowadays, with proper modification, CRIPSR/Cas9 has become a common gene editing tool, being able to cut the target gene with gRNA design.</p>
+                        <p>When it comes to artificial design of CRISPR/Cas9 system, how to sucessfully achieve recruitment of Cas9 depended on the structure of gRNA. In general, gRNA consists of 2 main parts, the former part is about 20 bp RNA sequences, which is exactly complementary to target genes, and it will be more impressive if these complementary sequence is at the beginning of the transcrption sites of gene. And the latter part is a secondary structure part which finally forms as hairpin used to recruit Cas9. During engineered section, this structual part can directly import from iGEM parts and what we need to take care is the fragment complementary to efflux system gene.</p>
+                        <p>CRISPR/Cas9 System for AcrB:</p>
+                        <figure><img src="https://static.igem.org/mediawiki/2015/4/49/CRISPR_Cas9_System_for_AcrB.png" alt="Figure 7:CRISPR/Cas9 System for AcrB "><figcaption>Figure 7:CRISPR/Cas9 System for AcrB</figcaption></figure>
+                        <p>CRISPR/Cas9 System for EmrE:</p>
+                        <figure><img src="https://static.igem.org/mediawiki/2015/f/fc/CRISPR_Cas9_System_for_EmrE.png" alt="Figure 8: CRISPR/Cas9 System for EmrE"><figcaption>Figure 8: CRISPR/Cas9 System for EmrE</figcaption></figure>
+                        <div class="divider"></div>
+                        <h4 id="adhesion-module" class="scrollspy">Adhesion Module</h3>
+                        <p><strong>What Material we Implement?-Theoretical Analysis</strong></p>
+                        <p>In order to produce slight deformation on the polymer film, we need to find a proper material to make it. According to our modeling, see more in <a href="http://tower.im">Modeling-Film Candidate</a>, pressure caused by adhesive bacteria is about 10^-4 Pa. Consequently, the wavelength of laser about 650nm is able to detect um-scale deformation. Plus considering cm squared film, we need the Young's Modulus of the film is less than 1GPa. </p>
+                        <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/4/4a/Ustc-filmcandidate.png">
+                              <figcaption>
+                              Film Candidate, from Wikipedia
+                              </figcaption>
+                             </figure>
+                        <p>The Young's Modulus of polypropylene is about 1.5Gpa, so it is selected as our candidate polymer film.</p>
+                        <p><strong>How to Achieve Stable Adhesion?</strong></p>
+                        <p>Bacteria adhering to polymer film is one of the most important parts in CACCI. In order to make bacteria bind tightly, we provide two possible and promising solutions to figure it out, one of which is coating bacteria with polylysine(PLL) and the other is to chemically covalent bind between bacteria and polymer film.</p>
+                        <p><strong><em>Strategy I: Coated with Polylysine</em></strong></p>
+                        <p>Polylysine refers to lysine homopolymers, whose precursor amino acid lysine is composed of two amino groups, one at the α-carbon and one at the ε-carbon. Either can be the location of polymerization, resulting in α-polylysine or ε-polylysine.</p>
+                        <figure><img src="https://static.igem.org/mediawiki/2015/3/33/Ustc-polylysine.png" alt="Figure 10: Polylysine"><figcaption>Figure 10: Polylysine</figcaption></figure>
+                        <p>α-Polylysine, a synthetic polymer, is composed of either L-lysine or D-lysine. "L" and "D" refer to the chirality at lysine's central carbon. This results in poly-L-lysine (PLL) and poly-D-lysine (PDL) respectively.</p>
+                        <figure><img src="https://static.igem.org/mediawiki/2015/8/85/Ustc-polylysine_interaction_with_cell.png" alt="Figure 11: The reaction of polylysine"><figcaption>Figure 11: The reaction of polylysine</figcaption></figure>
+                        <p>As a matter of fact, α-Polylysine is commonly used to coat tissue cultureware as an attachment factor which improves cell adherence. This phenomenon is based on the interaction between the positively charged polymer and negatively charged cells or proteins. This polycationic treatment on bacteria has already been proved with successful results when conducting microscopy experiment previously. </p>
+                        <figure><img src="https://static.igem.org/mediawiki/2015/1/10/Ustc-poly.png" alt="Figure 12: The adhesion of bacterial to polylysine "><figcaption>Figure 12: The adhesion of bacterial to polylysine</figcaption></figure>
+                        <p>As for <em>E. coli</em>, a gram-negative bacterium, whose surface is formed by lipopolysaccharides(LPP), which is negatively charged at physiological pH. The polymer chain of polylysine is positively charged and remains in stochastic globule conformation at pH under 12.0. Thus, polylysine would readily absorb to film we designed and gives a smooth coverage in a form of monolayer, containing tightly packed globule with average lateral dimension of 20 nm. Polylysine is biocompatible and should not affect bacteria. To get more information on our adhesion assay, please refer to <a href="http://tower.im">Results-Adhesion assay</a></p>
+                        <p><strong><em>Strategy II: Covalent Adhesion between bacterial membrane and film with proper modification.</em></strong></p>
+                        <p>To achieve covalently adhesion, we decided to implement the parts from <em>2014 iGEM TU_Eindhoven</em>, and the whole system including these parts:</p>
+                        <ul>
+                             <li>
+                                <p>DBCO-PEG4-NHS Ester (Dibenocyclooctyne-Polyethylene glycol-N-Hydroxysuccinimide), which covalently binding to polymer film.</p>
+                                    <figure><img src="https://static.igem.org/mediawiki/2015/9/9c/Chemotaxis.PNG" alt="Figure 13: DBCO"><figcaption>Figure 13: DBCO</figcaption></figure>
+                             </li>
+                             <li>
+                                <p>pAzF(p-Azido-L-Phenylalanine), a rare amino acid that would be implemented in bacterial transmembrane protein biosynthesis.</p>
+                                <figure><img src="https://static.igem.org/mediawiki/2015/c/cb/Dbco-ustc.png" alt="Figure 14: pAzF"><figcaption>Figure 14: pAzF</figcaption></figure>
+                             </li>
+                            <li>
+                                <p>In order to introduce pAzF into bacterial transmembrane protein, we need to mutate one of transmembrane protein, which is called COMPX. COMPX is able to mutate with a special tRNA. The plasmid containing tRNA is as following:</p>
+                                <figure><img src="https://static.igem.org/mediawiki/2015/0/04/Ustc-1436688284x-1376436670.png" alt="Figure 15: tRNA plasmid"><figcaption>Figure 15: tRNA plasmid</figcaption></figure>
+                             </li>
+                        </ul>
+                        <ul>
+                            <li>Covalent binding between DBCO and pAzF goes as following:
+                            <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/2/21/Ustc-1436688789x-1376436664.png">
+                              <figcaption>
+                              Figure 16:Schematic covalent interaction, this picture is extracted from guidebook of 2014 iGEM TU_Eindhoven
+                              </figcaption>
+                            </figure>
+                             </li>
+                        </ul>
+                        <p>To see more about constitution of our adhesion system, please refer to <a href="http://tower.im">Notebook-Protocol-Adhesion Preparation</a></p>
+                        <p><strong>Mutation of Transmembrane Protein OMPx in <em>E. coli</em></strong></p>
+                        <p>OMPx is a integral outer membrane protein X from  <em>Escherichia coli</em>, belonging to a family of highly conserved bacterial protein that promote bacterial adhesion to and entry into mammalian cells. Besides, these proteins as well play an important role in the resistance against attack by human complement system.</p>
+                        <p> The modified COMPX structure is illustrated as below:</p>
+                        <figure><img src="https://static.igem.org/mediawiki/2015/5/53/USTC-1437880487x-954497725.png" alt="Figure 17: The modified COMPX structure  "><figcaption>Figure 17: The modified COMPX structure</figcaption></figure>
+                        <p> As planned, OMPx will be mutated at residue 16, from glycine to p-Azido-L-Phenylalanine, zooming in the mutated site in orange:</p>
+                        <figure><img src="https://static.igem.org/mediawiki/2015/a/a5/USTC-1436688800x-1376436664.png" alt="Figure 18: The mutation site"><figcaption>Figure 18: The mutation site</figcaption></figure>
+                        <p><strong>The Covalent Reaction Model</strong></p>
+                        <p>Consequently, the model of adhesion goes like this: modified bacteria containing special p-Azido-L-Phenylalanine transformed tRNA carry OMPx with mutated p-Azido-L-Phenylalanine site. The laminated side in pAzF will covalently bind to alkenyl in DBCO-PEG4-NHS ester, and consequently undergo chemical reactions.</p>
+                        <figure><img src="https://static.igem.org/mediawiki/2015/0/0e/USTC-1437880652x-954497725.png" alt="Figure 19:The adhesion of bacterial to polymere"><figcaption>Figure 19:The adhesion of bacterial to polymere</figcaption></figure>
+                        <div class="divider"></div>
+                        <h4 id="package-your-cacci" class="scrollspy">Package Your CACCI</h4>
+                        <p>This is a collaboration project between BIT. More information on our collaboration, see <a href="http://tower.im">Collaboration</a>. We really appreciate BIT for their energetic spirits and friendly.</p>
+                        <p>Now, it's time to introduce why we need to package CACCI.</p>
+                        <p>Because CACCI has been engineered in an integrated way, it contains both chemotaxis improvement and permeability modification. If you want to construct CACCI with strategy II, it requires one more plasmid in CACCI, which is obviously quite difficult for other iGEM team to extract plasmids from parts and transform them again. Consequently, we USTC decided to package our CACCI and share to everyone who need them. </p>
+                        <p>Mechanism of packaging CACCI is quite simple, we firstly culture CACCI in LB liquid medium. (Attention: the LB liquid medium should contain glycerol which is used for cryopreservation. When bacteria grow in exponential phase. Get CACCI and culture them in skim milk containing sodium chloride. And we preserve those CACCI in refrigerator at minus 80 degree celcius for about 8 hours. Then put the CACCI into vacuum dryer, we would get CACCI in powder, ready for your recovery.</p>
+                        <p>More details on how to package CACCI please visit: <a href="http://tower.im">Protocol-Package your CACCI</a>. See our results at <a href="http://tower.im">Achievements-Package your CACCI</a></p>
+                        <div class="divider"></div>
+                        <h4 id="bibliography" class="scrollspy">Bibliography</h4>
+                        <ol>
+                            <li>
+                                <p>Comparative studies of bacteria with an atomic force microscopy operating in different modes. <strong>Ultramicroscopy 86 (2001) 121–128</strong> A.V. Bolshakova, I.V. Yaminsky.</p>
+                            </li>
+                            <li>
+                                <p>Measuring interfacial and adhesion forces between bacteria and mineral surfaces with
+                                    biological force microscopy. <strong>PII S0016-7037(00)00430-0</strong> Steven K. Lower, Michael F. hochella, JR.</p>
+                            </li>
+                        </ol>
                          </div>
                      </div>
@@ Line 159: / Line 374: @@
                              <ul class="section table-of-contents">
                                  <li>
-                                     <a href="#what-is-antibiotics-">What is Antibiotics? </a>
+                                     <a href="#who-is-cacci">Who is CACCI?</a>
                                  </li>
-                                <div class="divider"></div>
                                  <li>
-                                     <a href="#antimicrobial-resistance-and-abuses">Antimicrobial Resistance <br/>and Abuses</a>
+                                     <a href="#chemotaxis-modification-module">Chemotaxis Modification Module</a>
                                  </li>
-                                <div class="divider"></div>
                                  <li>
-                                     <a href="#antibiotics-production-and-contamination-in-china">Antibiotics Production <br/>and Contamination in China</a>
+                                     <a href="#integrative-modification-on-bacterial-permeability">Integrative Modification on Bacterial Permeability</a>
-                                 </li>
+                                </li>
+                                <li>
+                                    <a href="#adhesion-module">Adhesion Module</a>
+                                </li>
+                                <li>
+                                    <a href="#package-your-cacci">Package Your CACCI</a>
+                                </li>
+                                <li>
+                                    <a href="#bibliography">Bibliography</a>
+                                 </li>
                              </ul>
                          </div>
@@ Line 175: / Line 397: @@
              </div>
          </div>
-         <div id="ROSE" class="row">
+         <div id="SPRING" class="row">
              <div class="card hoverable">
                  <div class="row">
                      <div class="col s12 m9">
                          <div class="card-content">
+                            <h4 id="what-s-spring-" class="scrollspy">What's SPRING ?</h4>
-                            <h2 id="ROSEwhatis" class="scrollspy">What is ROSE?</h2>
-                             <p>
-                                <strong><em>Reporter Operater with Sensing Engine</em></strong>, in brief-<strong>ROSE</strong>, is a systematically modified bactieral machine for antibiotic concentration determination. Because antibiotic substances contamination occurs in very low concentration, such as ~2000ng/L or 2ug/L that is much lower than normal contaminants concentration, accurate detection by bactiera using simple genetic circuit. Consequently, ROSE is established with both genetically amplified circuit and systematic permeability reconstruction.
+                             <p>Spring, which is named after its inventor, is CACCI's sidekick. He helps CACCI in three
-                            </p>
+                                aspects, which are the optical aspect, the hardware aspect and the software aspect.
-                            <br />
+                                Using Michelson Interferometer, the optical part, Spring transforms the shape change of
-                             <p class="divider"></p>
+                                CACCI's membrane into the change of interference fringe. Spring's hardware, based on
-                            <br />
+                                Raspberry–Pi, allows spring to take photos or videos of the fringe. At last, the
-                            <h3 id="ROSESystematicOverviewofROSE" class="scrollspy">Systematic Overview of ROSE</h3>
+                                software analyses them and transmits the results to PC. CACCI depends on Spring.</p>
-                             <div><img src="https://static.igem.org/mediawiki/2015/5/5b/Overview_of_ROSE.png" alt="Overview of ROSE" /></div>
-                             <p>
+                             <p>Actually, SPRING is a measurement system which is designed to detect antibiotics in
-                                In general, ROSE as a community, containing two kinds of bactiera, among which Bactiera I specfically receives antibiotic concentration information from <em>antibiotic substance sensing promoters</em>, which were previously selected by Shimshon Belkin and Sahar Melamed in 2012. Then Bacteria I output information with LuxI, part of genetic expression regulator by quorum sensing, whose information is provided with positive amplification for Bacteria II. Bacteria II itself will automatically produce another part of regulator LuxR triggered by IPTG. Besides Bacteria II has a logically amlification circuit regulated by LuxI-LuxR complex. In this circuit containing two promoter regulaing two protein. The former one is regulated by Lux complex and produces cI inhibition protein and the latter one will be inhibited by cI protein and EGFP will be expressed when lack of cI protein. Consequently, this genetic amplification circuit, compare with others, will not directly enhance the intensity of GFP expression but to increase the signal resolution of concentration variation.
+                                natural water bodies. As figure-1 shows, the system SPRING includes Optical part,
-                            </p>
+                                Hardware part and Software part. Optical part is actually Michelson Interferometer, which
-                             <br />
+                                can transform the shape change of a film into the change of interference fringe. Based
-                             <p>
+                                on a Raspberry-Pi, hardware part can take photos or videos of interference fringe, then
-                                The design intention on bactieral membrane permeability improvement is directly from part of mechanism of antibiotics resistance. Theoratically, bacteria won't receive antibiotic substance bacause of mutation of its transmembrane protein and efflux system. Thus, we are attempting to modify bactiera in reverse way, that is overexpression of transmembrane protein, or more specifically porin, and inhibition of efflux system production through CRISPR.
+                                our software analyses them or transmits to your PC. Finally, we’ll see the results on
-                            </p>
+                                the screen. </p>
-                            <br />
-                            <p class="divider"></p>
+                             <figure><img src="https://static.igem.org/mediawiki/2015/8/8e/Ustc-spring1.png" alt="figure 1:The whole design of our electhic part and optical part "><figcaption>figure 1:The whole design of our electhic part and optical part</figcaption></figure>
-                            <br />
-                             <h3 id="ROSEAntibioticSubstanceSensingModule" class="scrollspy">Antibiotic Substance Sensing Module</h3>
+                             <p><em>Why SPRING?</em> Because we strongly call for limpid rives, just like spring flowing
-                             <p>
+                                between mountains. Nowadays, contamination issue in river becomes more and more serious.
-                                 Previous research from Sahar Melamed and Shimshon Belkin has already selected dozens of promoters responding to antibiotic concentration specificallly through the fluorescence signal.
+                                Not only excessive antibiotic emission in China, lots of other toxic heavy metal ion,
-                            </p>
+                                organic materials are found in river, which tremendously threaten public health and
-                             <p><img src="https://static.igem.org/mediawiki/2015/7/73/Promoter_Selection_with_Fluroescence_Intensity.png" alt="Promoter Selection with Fluroescence Intensity, *2012 Shimshon Belkin* " /></p>
+                                ecological balance. Consequently, we name our main body of device, SPRING, we hope with
-                            <p>
+                                the assistance of our SPRING, we are able to share and enjoy those clean river, like
-                                For instance, <em>micF</em> has relatively specific signal response when treated with sulfamethoxazole(255.2), sulfadimethoxine(44.9) and colistin(36.9), <em>soxS</em> is able to sense tetracycline(26.5) and oxytetracycline(26.1) at relative high intense. Besides, <em>recA</em> will effectively detect the existence of nalidixic acid and etc. These maximal induction of each promoter strain is tested in the course of 10h of exposure.
+                                those past days.</p>
-                            </p>
-                            <br />
+                             <h4 id="michelson-interferometer" class="scrollspy">Michelson Interferometer</h4>
-                             <p>
-                                The mechanism of these promoters are different from each other. However, basically, mechanisms consist of: - micF, an antisense ncRNA targeting ompF RNA, main endogenous porin of <em>Escherichia coli</em>, participating in post-transcriptionally regulation of this outer membrane protein F(ompF). micF is also regarded as one of the promoter of soxRS-regulated gene.
+                             <p>The Michelson interferometer is common configuration for optical interferometry and was
-                                <br />
+                                invented by Albert Abraham Michelson. Using a beam-splitter, a light source is split
-                                <img src="https://static.igem.org/mediawiki/2015/2/22/Antisense_ncRNA_with_ompF_mRNA_5%27_UTR.png" alt="micF: antisense ncRNA with ompF mRNA 5' UTR,*2011 JMB*" />
+                                into two arms. Each of those is reflected back toward the beam-splitter which then
-                             </p>
+                                combines their amplitudes interferometrically. The resulting interference pattern that
-                             <ul>
+                                is not directed back toward the source is typically directed to some type of
-                                 <li>
+                                photoelectric detector or camera. Depending on the interferometer's particular
-                                    SoxS, a transcriptional activator of oxidative stress genes in <em>E. coli</em>. SoxS <em>in vitro</em> binds the promoter of soxRS-regulated genes such as micF, sodA and recruits RNA polymerase to the promoters.
+                                application, the two paths may be of different lengths or include optical materials
-                                 </li>
+                                or components under test.<br>This is our design of optical part.</p>
-                            </ul>
+                                <figure><img src="https://static.igem.org/mediawiki/2015/0/06/Make19.jpg"><figcaption>
-                             <p>
+                                        Figure 2: The design of our Michelson Interferometer</figcaption></figure>
-                                In accordance with relative effective response of antibiotics, we are determined to implement micF to sense the existence of sulfamethoxazole and sulfadimethoxine in water sample,
-                                <img src="https://static.igem.org/mediawiki/2015/a/a1/Micf.png" alt="图片名称" /></p>
+                             <h4 id="hardware-module" class="scrollspy">Hardware Module</h4>
-                             <p>
-                                 and SoxS to detect the concentration of tetracycline or oxytetracycline.
+                             <p>Cooperating with the optical part, the electric circuit part makes use of a CCD camera to
-                                <img src="https://static.igem.org/mediawiki/2015/b/b4/Soxs.png" alt="图片名称" /></p>
+                                 take pictures of interference fringe, and then our software in the Raspberry-Pi analyses
-                             <p>In the circuit, micF and SoxS won't directly reporter fluorescence signal, instead, information will deliver through quorum sensing for positive amplification. In antibiotic substance sensing module, LuxI will be expressed directly for Bacteria II processing.</p>
+                                them and display the results on the screen.</p>
-                            <br />
-                             <p class="divider"></p>
+                             <figure>This is our design of hardware part.<br><img
-                            <br />
+                                    src="https://static.igem.org/mediawiki/2015/8/81/Ustc-spring3.png"
-                            <h3 id="ROSEQuorumSensingModule" class="scrollspy">Quorum Sensing Module</h3>
+                                    alt="Figure 3 : The design of our hardware, looing from outside"><figcaption>Figure 3 : The design of our hardware, looing from outside</figcaption></figure>
-                             <p>
-                                Using community with different compartments would further improve the signal amplification, as known, quorum sensing is a system for stimulation(some signaling molecules called autoinducers) and response(receptor that will transcrpt certain genes) that is related to the bactieral concentration in the system. Many gene expression is naturally regualted by quorum sensing, which is also treated as social interation in bacterial community.
+                            <p>This is the SPRING we built. </p>
-                                <img src="https://static.igem.org/mediawiki/2015/0/0d/Che.png" alt="图片名称" />
-                            </p>
+                             <figure><img src="https://static.igem.org/mediawiki/2015/b/ba/Make13.jpg"
-                            <br />
+                                    alt="Figure 4: The real SPRING we built"><figcaption>Figure 4: The real SPRING we built</figcaption></figure>
-                             <p>
-                                In some gram-negative bacteria, such as <em>Vibro fischeri</em>, biosynthesis of autoinducers is used for yield of bioluminescence through luciferase. The signaling molecule used by <em>V. fischeri</em> is an acylated homoserine lactone(AHL),called N-(3-oxohexanol)-homoserine lactone, which can be engineered into <em>E. coli</em>. The signaling molecule will be produced in the cytoplasm using LuxI synthease enzyme and then secreted through the cell membrane to the extracelluar surroundings. When N-(3-oxohexanol)-homoserine lactone begins diffusing back into cells or other bacteria, LuxR will recognize the existence of N-(3-oxohexanol)-homoserine lactone with a threshold concentration, about 10ug/mL. Finally, target gene such as LuxlCDABE will be activated with LuxR and N-(3-oxohexanol)-homoserine lactone complex.
-                            </p>
-                            <br />
+                             <h4 id="electric-circuit" class="scrollspy">Electric Circuit</h4>
-                            <p>
-                                 It has been proved that LuxR has evolutionary conservation in many gram-negative bacteria, which enables <em>E. coli</em> to receive AHL from other bacteria. Thus we engineer Bacteria I to release quorum sensing molecules to Bacteria II for exponentailly amplified results.
+                             <p>The Raspberry Pi is a low cost, credit-card sized computer that plugs into a computer
-                            </p>
+                                 monitor or TV, and uses a standard keyboard and mouse. It is a capable little device
-                             <br />
+                                that enables people of all ages to explore computing, and to learn how to program in
-                             <p>
+                                languages like Scratch and Python. It’s capable of doing everything you’d expect a
-                                <img src="https://static.igem.org/mediawiki/2015/f/f3/RBS.png" alt="图片名称" />
+                                desktop computer to do, from browsing the internet and playing high-definition video, to
-                                 To achieve antibiotic concentration corresponding to AHL production, AHL produced by Bacteria I will be regulated by antibiotic substance sensing promoters. And in Bacteria II, LuxR will be directly expressed regulated by lac operon, triggered by IPTG. With treatment of IPTG, LuxR will be consitutively expressed to ensure LuxR-AHL complex will intiate the expression of required genes.
+                                making spreadsheets, word-processing, and playing games. The module we used is
-                            </p>
+                                 Raspberry-PI 2B.</p>
-                             <br />
-                            <p class="divider"></p>
+                             <figure><img src="https://static.igem.org/mediawiki/2015/b/b9/Make20.jpg"
-                            <br />
+                                   alt="Figure 5: The real electric part we built" ><figcaption>Figure 5: The real electric part we built</figcaption></figure>
-                            <h3 id="ROSESecondLevelAmplificationModule" class="scrollspy">Second Level Amplification Module</h3>
-                             <p>
+                             <p>As the picture shows, the battery powers the little fan and the laser part. The little
-                                 In addition of signal amplification through quorum sensing, an artificial genetically engineered cascade magnification circuit is also implemented in ROSE construction.
+                                 fan is necessary because of the need to lower the temperature of the board. </p>
-                            </p>
-                             <p>
+                            <figure><img src="https://static.igem.org/mediawiki/2015/e/e0/Ustc-myo4.png"
-                                <img src="https://static.igem.org/mediawiki/2015/2/25/Cl.png" alt="图片名称" />
+                                    alt="Figure 6: The electrical source and the transformer"><figcaption>Figure 6: The electrical source and the transformer</figcaption></figure>
-                            </p>
-                             <p>
+                             <p>The transformer transforms 12 volts to 5 volts, and the breadboard supplies power for the
-                                 In this cascade signal circuit, LuxR-AHL complex trigger the activation of promoter Lux, then express cI derived from λ phage. Since cI is a highly effcient repressor that is a low concentration of cI will completely repress the effiency of λ promoter. As a result, EGFP will be drastically and negatively regulated when treated with antibiotic substances.
+                                laser and the fan. </p>
-                            </p>
-                            <br />
+                             <p>A breadboard is a construction base for prototyping of electronics. Originally it was
-                            <p>
+                                literally a bread board, a polished piece of wood used for slicing bread. In the 1970s
-                                 It is notable that this second level amplication module won't directly expand the signal intensity, in another way, it will indirectly enlarge the resolution of different antibiotic concentration gradients. Our theoratical modeling also illustrate a higher resolution implementing this amplification circuit.
+                                the solderless breadboard (AKA plugboard, a terminal array board) became available and
-                            </p>
+                                nowadays the term "breadboard" is commonly used to refer to these. "Breadboard" is also
-                             <br />
+                                a synonym for "prototype".</p>
-                            <p class="divider"></p>
-                            <br />
+                             <figure>Then let's see the screen , keyboard and mouse.<br><img
-                             <h3 id="ROSEIntegrativeModificationonBacterialPermeability" class="scrollspy">Integrative Modification on Bacterial Permeability</h3>
+                                    src="https://static.igem.org/mediawiki/2015/8/8a/Make23.jpg"
-                             <p>
+                                    alt="Figure 7: screen , keyboard and mouse"><figcaption>Figure 7: screen , keyboard and mouse</figcaption></figure>
-                                 Our ROSE construction is systematically not only include quorum sensing and logic amplication, but containing some permeability improvement by inhibition of efflux system and overexpression of exogenous porin. Nonpolar residues face outward so as to interact with the nonpolar lipid membrane, whereas the polar residues face inwards into the center of the beta barrel to interact with the aqueous channel.
-                            </p>
+                             <p>The screen, keyboard and the mouse are connected to the RaspberryPi via USB.</p>
-                             <br />
-                            <p>
+                            <p>We use CCD camera to capture images.</p>
-                                <strong>Porin: OprF from <em>Pseudomonus aeruginosa</em></strong> Porins are beta barrel proteins crossing a celluar membrane and act as a pore through which molecules are able to diffuse.
+                            <figure><img
-                            </p>
+                                    src="https://static.igem.org/mediawiki/2015/d/dc/Ustc-myo5.png"
-                            <br />
+                                    alt="Figure 8: CCD camera "><figcaption>Figure 8: CCD camera</figcaption></figure>
-                             <p>
+                                    <p>A charge-coupled device (CCD) is a device for the
-                                In <em>E. coli</em> different porins, for example OmpF and OmpC is expressed endogenously in different situations for different molecules. But these natural occurring porines are only permeable for molecules like sugars smaller than 600 Da. And more importantly, implement of micF, an antisense ncRNA may inhibit the material diffusion through OmpF. Therefore, it is necessary to import an exogenous porin with higher effiency of small molecular tranportation, such as OprF derived from <em>Pseudomonas aeruginosa</em>.
+                                 movement of electrical charge, usually from within the device to an area where the
-                                <img src="https://static.igem.org/mediawiki/2015/e/ed/Pseudomonas_aeruginosa.png" alt="图片名称" />
+                                charge can be manipulated, for example conversion into a digital value. This is achieved
-                            </p>
+                                by "shifting" the signals between stages within the device one at a time. CCDs move
-                            <br />
+                                charge between capacitive bins in the device, with the shift allowing for the transfer
-                             <p>
+                                of charge between bins.</p>
-                                OprF represents one of the largest pore sizes on bacterial outer membranes, allowing diffusion of polysaccharides in a range of 2000 to 3000 Da. The overexpression of OprF in <em>E. coli</em> will drastically improve the antibiotic absorption capability, like antibiotic condensation occured in <em>E. coli</em>.
-                            </p>
+                             <h4 id="optical-part-michelson-interferometer" class="scrollspy">Optical Part: Michelson Interferometer</h4>
-                             <br />
-                            <p>
+                             <figure><img src="https://static.igem.org/mediawiki/2015/1/1a/Make24.jpg"
-                                <strong>Viroporin: SCVE from SARS Virus</strong> Viroporins are similar to porins in that they oligomerize within the membrane to form pores and this oligomerization often occurs between hydrophobic, transmembrane and alpha-helical domains of the proteins.</p>
+                                    alt="Figure 9: 2nd Michelson Interferometer "><figcaption>Figure 9: 2nd Michelson Interferometer</figcaption></figure>
-                            <p><img src="https://static.igem.org/mediawiki/2015/2/29/Proteins1.jpg" alt="图片名称" />
-                             </p>
+                            <p>The Michelson interferometer is common configuration for optical interferometry and was
-                             <p>
+                                 invented by Albert Abraham Michelson. Using a beam-splitter, a light source is split
-                                SCVE, SARS Caronavirus Envolope protein, is a kind of effective viroporin within 76 residues that is found normally pentamerized in bacteria. SCVE is permeable for many small molecules, which is originately designed for apoptosis of cells triggered by virus. Implementation of SCVE in ROSE may potentially improve the permatilibty of small molecules including antibiotics.
+                                into two arms. Each of those is reflected back toward the beam-splitter which then
-                            </p>
+                                combines their amplitudes interferometrically. The resulting interference pattern that
-                            <br />
+                                is not directed back toward the source is typically directed to some type of
-                             <p>
+                                photoelectric detector or camera. Depending on the interferometer's particular
-                                <strong>Inhibition of Efflux System using CRIPSR</strong> Efflux system is a kind of mechanism found in microbes responsible for many moving molecules, especially some toxic substances, microbiotics.
+                                application, the two paths may be of different lengths or include optical materials or
-                                <img src="https://static.igem.org/mediawiki/2015/f/f6/Fmicb-04-00007-g001.jpg" alt="Strucutual Model of AcrAB System" />
+                                components under test.</p>
-                                <img src="https://static.igem.org/mediawiki/2015/d/d2/Strucuture_of_EmrE%2C_3B61_in_PDB.png" alt="Strucuture of EmrE, 3B61 in PDB" />
-                             </p>
+                             <figure><img src="https://static.igem.org/mediawiki/2015/1/10/Make12.jpg"
-                             <p>
+                                    alt="Figure 10: The real optical part we built"><figcaption>Figure 10: The real optical part we built</figcaption></figure>
-                                 According to previous research, efflux system contributes a lot for bacterial antibiotic resistance, or multidrug resistance(MDR). For example, in <em>E. coli</em>, AcrAB efflux system has a physiological role of pumping out many bile acids, fatty acids to lower their intracelluar toxicty. It would be an attemptation to go directly reverse to antibiotic resistance, that is to inhibit the function of efflux system. In order to completely inhibit the major efflux system of <em>E. coli</em> in genetic level, CRIPSR would be an impressive solution for totally deletion of the function of efflux system.
+                             <p>The following picture shows our
-                             </p>
+                                 details of optical part, which includes various splitters.</p>
-                             <br />
+                             <figure><img src="https://static.igem.org/mediawiki/2015/0/06/Make19.jpg"
-                             <p>
+                                        alt="Figure 11: 3rd Michelson Interferometer "><figcaption>Figure 11: 3rd Michelson Interferometer</figcaption></figure>
-                                 Clutersted reularly interspacaed short palindromic repeats, abbreviated as <em>CRIPSR</em>, are some fragements in prokayotic DNA with short repeats. Natually, CRISPR/Cas system is an important immune system that is able to resist the existence and reproduction of exogenous genetic materials, directly recognizing and then cut these genetic fragments, such as phages and plasmids, by CRIPSR spacers, which is quite similar to RNA interference(RNAi) discovered in eukaryotic organisms. Cas9, which is CRISPR Associated Protein 9 in brief, is an RNA-guding DNA endonuclease enzyme associated with the CRISPR segments derived naturally from * Streptococcus pyogenes* and other bacteria. It has been showed that Cas9 plays an important role in memorizing, interrogating and ultimately cleaving these foreign DNA. Mechanism of cleavage is to unwind the foreign DNA and check whether there is a about 20 bp fragments that is complementary to guide RNA (gRNA, in brief). When the recognition result turns to yes, Cas9 will cleave the foreign DNA. Nowadays, with proper modification, CRIPSR/Cas9 has become a common gene editing tool, being able to cut the target gene with gRNA design.
-                             </p>
+                             <p>Beam splitter combination, consists of three mirrors, a 50% reflection - 50% transmission
-                             <p><img src="https://static.igem.org/mediawiki/2015/9/9b/Crispr-caslg.jpg" alt="Schematic Model of CRISPR/Cas9 System" /></p>
+                                 beam splitter (50%(R)/50%(T) in brief), a 80% reflection - 20% transmission beam
-                             <p>
+                                splitter(80%(R)/20%(T) in brief) and a 10% reflection- 90% transmission beam
-                                When it comes to artificial design of CRISPR/Cas9 system, how to sucessfully achieve recruitment of Cas9 depends on the structure of gRNA. In general, gRNA consists of 2 main parts, the former part is about 20 bp RNA sequences, which is exactly complementary to target genes, and it will be more impressive if these complementary sequence is at the beginning of the transcrption sites of gene. And the latter part is a secondary structure part which finally forms as hairpin used to recruit Cas9. During engineered section, this structual part can directly import from iGEM parts and what we need to take care is the fragment complementary to efflux system gene.
+                                splitter(10%(R)/90%(T) in brief). 1)50%(R)/50%(T) is in the front of picture. The
-                            </p>
+                                reflection light will project to detecting film , while the transmission beam splitter
-                            <p>CRISPR/Cas9 System for AcrB:
+                                will continue going to reflection mirror. 2) Then why do we need The 80%(R)/20%(T),
-                                <img src="https://static.igem.org/mediawiki/2015/4/49/CRISPR_Cas9_System_for_AcrB.png" alt="图片名称" /></p>
+                                along with 10%(R)/90%(T)? Actually these combination is really important to decrease the
-                             <p>CRISPR/Cas9 System for EmrE:
+                                relatively high intensity reflected from reflector. Much higher intensity from reflector
-                                <img src="https://static.igem.org/mediawiki/2015/f/fc/CRISPR_Cas9_System_for_EmrE.png" alt="图片名称" /></p>
+                                 will not fit the formation condition of interference pattern.
-                            <br />
+%(R)/20%(T)+10%(R)/90%(T), approximately allowing 18% light approach reflector, are
-                            <p class="divider"></p>
+                                combined together on the bottom of the picture.</p>
-                            <br />
+                             <figure><img src="https://static.igem.org/mediawiki/2015/9/95/Ustc-myo6.png" alt="Figure 12: The beam splitter used in the michelson interferometer"><figcaption>Figure 12: The beam splitter used in the michelson interferometer</figcaption></figure>
-                             <h3 id="ROSETheoreticalAdvantage" class="scrollspy">Theoretical Advantage</h3>
-                             <p><img src="image/QQ%E6%88%AA%E5%9B%BE20150803214403.png" alt="图片名称" />
+                             <h4 id="mechanical-part" class="scrollspy">Mechanical part</h4>
-                                <img src="image/QQ%E6%88%AA%E5%9B%BE20150803214410.png" alt="图片名称" /></p>
-                            <br />
+                             <p>We use a kind of plastic called Acrylic to build our mechanical part--the BOX. Acrylic is
-                             <p class="divider"></p>
+                                 totally transparent while there is a piece of paper stuck on it. Our box was designed by
-                             <br />
+                                ourselves and it's processed by company from the website TAOBAO.</p>
-                            <h3 id="ROSEBibliography" class="scrollspy">Bibliography</h3>
+                             <figure><img src="https://static.igem.org/mediawiki/2015/3/35/Make14.jpg" alt="Figure 13: The Acrylic board we used "><figcaption>Figure 13: The Acrylic board we used</figcaption></figure>
-                            <p>
+                             <p>And we use the electric drill to cut it. The head of the drill can be replaced by many part such as saws or polish-head.</p>
-.<i>A bacterial reporter panel for the detection and classification of antibiotic substances <b>Microbial Biotechnology (2012) 5(4), 536-548</b>Sahar Melamed, Shimshon Belkin</i>
+                            <figure><img src="https://static.igem.org/mediawiki/2015/1/19/Make10.jpg" alt="Figure 14:The electric drill we used"><figcaption>Figure 14:The electric drill we used</figcaption></figure>
-                            </p>
+                             <p>Oh, all parts should be fixed with hot-melt adhesive (hot glue), in order to maintain stability. The glue is tacky when hot, and solidifies in a few seconds to one minute. Hot melt adhesives can also be applied by dipping or spraying.</p>
-                            <br />
+                             <figure><img src="https://static.igem.org/mediawiki/2015/c/cf/Make16.jpg" alt="Figure 15: One example of hot glue using"><figcaption>Figure 15: One example of hot glue using</figcaption></figure>
-                            <p>
-.<i>Signal-Amplifying Genetic Circuit Enables In Vivo Observation of WeakPromoter Activation in the Rhl Quorum Sensing System<b>Wiley InterScience 2005</b>DOI: 10.1002/bit.20371</i>
+                             <h4 id="software-and-fringe-analysis" class="scrollspy">Software and Fringe analysis</h4>
-                            </p>
-                            <br />
+                             <p>Get your pictures! For example, you'll see this one.</p>
-                             <p>
-.<i>MicF: An Antisense RNA Gene Involved in Response of Escherichia coli to Global Stress Factors <strong>2001 JMB</strong> doi:10.1006/jmbi.2001.5029</i>
+                             <figure><img src="https://static.igem.org/mediawiki/2015/9/98/20150823001.jpg"
+                                    alt="Figure 16:An example of pictures you'll get using the CCD camera"><figcaption>Figure 16:An example of pictures you'll get using the CCD camera</figcaption></figure>
+                            <p>And then analyze the picture. Wanna know how? Read on.</p>
+                            <figure><img src="https://static.igem.org/mediawiki/2015/c/c0/20150831001.png"
+                                    alt="Figure 17: How to measure--the deformation of film"><figcaption>Figure 17: How to measure--the deformation of film</figcaption></figure>
+                             <p>Methods</p>
+                             <p>(1)</p>
+                            <ol>
+                                 <li>Take a series of photos at the same position in a short time.</li>
+                                <li>Superpose these photos to sharp the edge of every object.</li>
+                                <li>Choose two points in multi-image, the point must be on the black fringes.</li>
+                                <li>Scanning these two fringes to find the shortest distance between them.</li>
+                                <li>Calculate the radius and rank of every fringes.</li>
+                                <li>Calculate the deformation of film.</li>
+                             </ol>
+                             <p>(2)</p>
+                             <ol>
+                                 <li>Take a series of photos at the same position in a short time.</li>
+                                <li>Superpose these photos to sharp the edge of every object.</li>
+                                <li>Calculate the number of fringes on vertical direction(y axis).</li>
+                                <li>Transform the number to deformation of the film.</li>
+                            </ol>
+                            <p>More details on coding please refer to :(Add super link here)</p>
+                            <p>This is our program, which was written in MATLAB. This program is used to simulate fringes
+                                pattern delivered by film I.</p>
+                            <p>Code:</p>
+ <div><textarea id="code" name="code">function drawndh(r,h,t,n) %r is radius of film，h is the distance of deformation，t is the drift angle of
+    film，n is pixel number
+    wl=6.5e-7; %wave length
+    R=r.^2/(2*h);
+    [x,y]=meshgrid(linspace(-0.004,0.004,n),linspace(-0.004,0.004,n));
+    r2=x.^2+y.^2;
+    delta=pi*r2/(R*wl)-2*pi*y*sin(t)/wl;
+    I=abs(sin(delta).^2);
+    imshow(I);
+    end
+</textarea></div>
+                             <p>Demo</p>
+                             <figure><img src="https://static.igem.org/mediawiki/2015/c/cd/0.02-5e-6-5e-4.jpg"
+                                            alt="Figure 18:: Film II deformation simulation result"><figcaption>Figure 18:: Film II deformation simulation result</figcaption></figure>
+                             <p>Using this code, we are able to capture some important parameters in Film I.</p>
+                            <p>Code</p>
+<div><textarea id="code1" name="code">function ms3=solvendh(X,nx,ny,n,dx,dy)%nx is the number of fringes on x axis,ny is the number of fringes on y
+    axis,n is the number of fringes on diag,dx is the x length of CCD,dx is the x length of CCD.
+    x=X(1);
+    y=X(2);
+    z=X(3);
+    l=1.2e-4;
+    ms3(1)=(2*(z+nx)*l)^0.5-(2*z*l)^0.5-((x+dx)^2+y^2)^0.5+(x^2+y^2)^0.5;
+    ms3(2)=(2*(z+ny)*l)^0.5-(2*z*l)^0.5-(x^2+(y+dy)^2)^0.5+(x^2+y^2)^0.5;
+    ms3(3)=(2*(z+n)*l)^0.5-(2*z*l)^0.5-((x+dx)^2+(y+dy)^2)^0.5+(x^2+y^2)^0.5;
+</textarea></div>
+                             <p>Demo</p>
+                            <figure><img src="https://static.igem.org/mediawiki/2015/0/0d/20150919071.png"
+                                            alt="Figure 19: Film I Fringes analysis running result"><figcaption>Figure 19: Film I Fringes analysis running result</figcaption></figure>
+                             <h4 id="cooperate-with-rose-and-cacci" class="scrollspy">Cooperate with ROSE and CACCI</h4>
+                             <p>Along with ROSE and CACCI , SPRING will help detect antibiotic as well.</p>
+                            <figure><img
+                                    src="https://static.igem.org/mediawiki/2015/7/73/Ustc-myo10.png"
+                                    alt="Figure 20: 4th Michelson Interferometer "><figcaption>Figure 20: 4th Michelson Interferometer</figcaption></figure>
+                             <p>For details , you can look at this picture.</p>
+                             <figure><img src="https://static.igem.org/mediawiki/2015/3/33/Make28.jpg"alt="Figure 21:The cover glass and bacteria"><figcaption>Figure 21:The cover glass and bacteria</figcaption></figure>
+                             <p>In order to
+                                 measure the nano distance, we need to adhere bacteria to a surface, the film we used is
+                                processed cover slip.
                              </p>
+                            <figure><img src="https://static.igem.org/mediawiki/2015/3/37/Make29.jpg"
+                                    alt="Figure 22: Interference will be observed"><figcaption>Figure 22: Interference will be observed</figcaption></figure>
+                            <p>During our experiment , when elastic deformation--caused by the movement of the bacteria
+                                containing cheZ parts--occurred on the film, interference will be observed.</p>
                          </div>
                      </div>
@@ Line 340: / Line 645: @@
                              <ul class="section table-of-contents">
                                  <li>
-                                     <a href="#ROSEwhatis">What is ROSE?</a>
+                                     <a href="#what-s-spring-">Who is SPRING?</a>
-                                 </li><div class="divider"></div>
+                                 </li>
                                  <li>
-                                     <a href="#ROSESystematicOverviewofROSE">Systematic Overview <br/>of ROSE</a>
+                                     <a href="#michelson-interferometer">Michelson Interferometer</a>
-                                 </li><div class="divider"></div>
+                                 </li>
                                  <li>
-                                     <a href="#ROSEAntibioticSubstanceSensingModule">Antibiotic Substance<br/> Sensing Module</a>
+                                     <a href="#hardware-module">Hardware Module</a>
-                                 </li><div class="divider"></div>
+                                 </li>
                                  <li>
-                                     <a href="#ROSEQuorumSensingModule">Quorum Sensing Module</a>
+                                     <a href="#electric-circuit">Electric Circuit</a>
-                                 </li><div class="divider"></div>
+                                 </li>
                                  <li>
-                                     <a href="#ROSESecondLevelAmplificationModule">Second Level<br/> Amplification Module</a>
+                                     <a href="#optical-part-michelson-interferometer">Optical Part: Michelson Interferometer</a>
-                                 </li><div class="divider"></div>
+                                 </li>
                                  <li>
-                                     <a href="#ROSEIntegrativeModificationonBacterialPermeability">Integrative Modification <br/>on Bacterial Permeability</a>
+                                     <a href="#mechanical-part">Mechanical part</a>
-                                 </li><div class="divider"></div>
+                                 </li>
                                  <li>
-                                     <a href="#ROSETheoreticalAdvantage">Theoretical Advantage</a>
+                                     <a href="#software-and-fringe-analysis">Software and Fringe analysis</a>
-                                 </li><div class="divider"></div>
+                                 </li>
                                  <li>
-                                     <a href="#ROSEBibliography">Bibliography</a>
+                                     <a href="#cooperate-with-rose-and-cacci">Cooperate with ROSE and CACCIs</a>
                                  </li>
                              </ul>
                          </div>
@@ Line 370: / Line 674: @@
              </div>
          </div>
-         <div id="CACCI" class="row">
+         <div id="ROSE" class="row">
              <div class="card hoverable">
                  <div class="row">
                      <div class="col s12 m9">
                          <div class="card-content">
-                             <h3 id="who-is-cacci-" class="scrollspy">Who is CACCI?</h3>
+                             <h4 id="ROSEwhatis" class="scrollspy">What is ROSE?</h4>
                              <p>
-                                 <strong><em>CACCI</em></strong>, called the same as Kathy, is a constructed bacteria for <strong><em>Characterization of Antibiotic Concentration based on Chemotaxis and Interference.</em></strong>
+                                 <strong><em>Reporter Operator with Sensing Engine</em></strong>, in
+                                brief-<strong>ROSE</strong>, is a systematically modified bacterial machine for
+                                antibiotic concentration determination. Because antibiotic substances contamination
+                                occurs in very low concentration, such as ~2000ng/L or 2ug/L that is much lower than
+                                normal contaminants concentration, accurate detection by bacteria using simple genetic
+                                circuit. Consequently, ROSE is established with both genetically amplified circuit and
+                                systematic permeability reconstruction.
                              </p>
-                             <br />
-                             <p>Although, construction of ROSE will specifically recognize the concentration of different antbiotics in the river samples, its feature is also deficiency: that is too specificity may hurt extensive measurement. Besides, using traditional genetically modified circuit to express report is still time-consuming, comparing with general physical or chemical measurement approaches.</p>
+                             <div class="divider"></div>
-                             <br />
+                             <h4 id="ROSESystematicOverviewofROSE" class="scrollspy">Systematic Overview of ROSE</h4>
-                            <p>Consequently, we orginally esatblished CACCI based on chemotaxis and interference to make a wider scale level antibiotic molecules detection, even other chemical molecules. So, the mechanism and construction of CACCI can be divided into the following schedules:</p>
-                            <br />
+                             <figure><img src="https://static.igem.org/mediawiki/2015/5/5b/Overview_of_ROSE.png"
-                            <ol>
+                                    alt="Overview of ROSE"/><figcaption>Overview of ROSE</figcaption></figure>
-                                <li><strong>Chemotaxis Modification</strong>: to increase the bacterial movement capability, modification based on <em>E. coli</em> chemotaxis related parts cheZ will be introduced into bacteria.</li>
-                                <li><strong>Adhesion</strong>: According to our modeling, we need to adhere bacteria to a surface. Theoratically, interference will be observed when elastic deformation occured on membrane caused by bacteria motility containing cheZ parts, covalent ligation will be established between bacteria and membrane to ensure the stable adhesion. In order to achieve covalently adhesion, modification on bacteria transmembrane protein will be necessary.</li>
+                            <p>In general, ROSE as a community, containing two kinds of bacteria, among which Bacteria I
-                                 <li><strong>Interference</strong>: When bacteria have been adhered to membrane and treated in water sample, movement will cause interference. The interference pattern will be different at distinct chemical molecules concentration.</li>
+                                 specifically receives antibiotic concentration information from <em>antibiotic substance
-                                 <li><strong>Processing</strong>: These patterns will be captured by CCD camera. Our control system containing interference pattern recognition will process these patterns, and finally a work curve will be established corresponding antibiotics concentration to different interference pattern. For friendly operation, graphic user interface(GUI) is established better for user utilization on this program.</li>
+                                    sensing promoters</em>, which were previously selected by Shimshon Belkin and Sahar
-                             </ol>
+                                Melamed in 2012. Then Bacteria I output information with LuxI, part of genetic
-                            <br />
+                                expression regulator by quorum sensing, whose information is provided with positive
-                             <p class="divider"></p>
+                                amplification for Bacteria II. Bacteria II itself will automatically produce another
-                             <br />
+                                part of regulator LuxR triggered by IPTG. Besides Bacteria II has a logically
-                            <h3 id="chemotaxis-modification-module" class="scrollspy">Chemotaxis Modification Module</h3>
+                                 amplification circuit regulated by LuxI-LuxR complex. In this circuit containing two
-                             <p><strong>What is Chemotaxis?</strong>
+                                promoter regulating two protein. The former one is regulated by Lux complex and produces
-                                 <br />
+                                cI inhibition protein and the latter one will be inhibited by cI protein and EGFP will
-                                 Chemotaxis is a phenomenon of the movement of an organism, specifically cells or bacteria, stimulated by chemical molecules. As for bacteria, naturally, chemoreceptors were expressed on bacteria membrane, which enables bacteria to 'sense' the existence of chemical molecule concentration in the surroundings. Chemotaxis consists of two situation, including positive chemotaxis and negative chemotaxis, which respectively demonstrate different behaviour when exposed to surroundings with chemical molecules beneficial or harmful to bactiera metabolism and reproduction.
+                                be expressed when lack of cI protein. Consequently, this genetic amplification circuit,
+                                compare with others, will not directly enhance the intensity of GFP expression but to
+                                increase the signal resolution of concentration variation.</p>
+                             <p>The design intention on bacterial membrane permeability improvement is directly from part
+                                of mechanism of antibiotics resistance. Theoretically, bacteria won't receive antibiotic
+                                substance because of mutation of its transmembrane protein and efflux system. Thus, we
+                                are attempting to modify bacteria in reverse way, that is overexpression of
+                                transmembrane protein, or more specifically porin, and inhibition of efflux system
+                                production through CRISPR.</p>
+                             <div class="divider"></div>
+                             <h4 id="ROSEAntibioticSubstanceSensingModule" class="scrollspy">Antibiotic Substance Sensing
+                                Module</h4>
+                             <p>
+                                 Previous research from Sahar Melamed and Shimshon Belkin has already selected dozens of
+                                 promoters responding to antibiotic concentration specifically through the fluorescence
+                                signal.
                              </p>
-                             <br />
-                             <p>The chemotaxis mechanism in <em>E. coli</em> can be illstustrated as following:</p>
+                             <figure>
-                             <p><img src="image/Chemotaxis.PNG" alt="Mechanism of Chemotaxis, from WITS-CSIR_SA 2011 iGEM"></p>
+                              <img src="https://static.igem.org/mediawiki/2015/7/73/Promoter_Selection_with_Fluroescence_Intensity.png">
+                              <figcaption>
+                              Promoter Selection with Fluroescence Intensity, *2012 Shimshon Belkin*
+                              </figcaption>
+                            </figure>
+                            <p>For instance, <em>micF</em> has relatively specific signal response when treated with
+                                sulfamethoxazole(255.2), sulfadimethoxine(44.9) and colistin(36.9), <em>soxS</em> is
+                                able to sense tetracycline(26.5) and oxytetracycline(26.1) at relative high intense.
+                                Besides, <em>recA</em> will effectively detect the existence of nalidixic acid and etc.
+                                These maximal induction of each promoter strain is tested in the course of 10h of
+                                exposure.</p>
+                             <p>The mechanism of these promoters are different from each other. However, basically,
+                                mechanisms consist of: - micF, an antisense ncRNA targeting ompF RNA, main endogenous
+                                porin of <em>Escherichia coli</em>, participating in post-transcriptionally regulation
+                                of this outer membrane protein F(ompF). micF is also regarded as one of the promoter of
+                                soxRS-regulated gene.</p>
+                             <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/2/22/Antisense_ncRNA_with_ompF_mRNA_5%27_UTR.png">
+                              <figcaption>
+                              micF: antisense ncRNA with ompF mRNA 5' UTR,*2011 JMB*
+                              </figcaption>
+                            </figure>
                              <ul>
-                                 <li>
+                                 <li>SoxS, a transcriptional activator of oxidative stress genes in <em>E. coli</em>.
-                                    <p>When absence of chemical, there is no molecule binds to chemorecptor, cheW, an intracellular protein, is associated with cheA and cheA will autophosphorylate itself and capture phosphate group from ATP. Later, phosphate will be transferred to cheY and the phosphorylated cheY associates with the flagellar motors making flagellum rotates clockwise. In general, bacteria tumble in the absence of chemical.</p>
+                                    SoxS <em>in vitro</em> binds the promoter of soxRS-regulated genes such as micF,
-                                </li>
+                                    sodA and recruits RNA polymerase to the promoters.
-                                <li>
-                                    <p>When presence of chemicals, cheA is free from cheW interation, and cheZ will efficiently remove phosphate group from cheY. Those dephosphorylated cheY cannot bind with flagellar motors, and consequently, flagellar rotats counter-clock which can be observed as bacteria swim directly. In some special situation, cheR will methylates chemoreceptors to remain the disasscoation between cheA and cheW. Then, bacteria will still swim without tumbles.</p>
                                  </li>
                              </ul>
-                             <p><strong>Genetically Engineered Chemotaxis Bacteria</strong>
+                             <p>In accordance with relative effective response of antibiotics, we are determined to
-                                <br>Because of lack of natural chemoceptor for antibiotics, we need to construct a genetic circuit specifically to sensing antibiotics, and according to the mechanism of chemotaxis, we need to overexpress cheZ to improve its phosphorlation capability on cheY. The model is illstruated as following:
+                                implement micF to sense the existence of sulfamethoxazole and sulfadimethoxine in water
+                                sample,</p>
+                            <p><img src="https://static.igem.org/mediawiki/2015/a/a1/Micf.png"/></p>
+                            <p>and SoxS to detect the concentration of tetracycline or oxytetracycline.</p>
+                            <p><img src="https://static.igem.org/mediawiki/2015/b/b4/Soxs.png"/></p>
+                            <p>In the circuit, micF and SoxS won't directly reporter fluorescence signal, instead,
+                                information will deliver through quorum sensing for positive amplification. In
+                                antibiotic substance sensing module, LuxI will be expressed directly for Bacteria II
+                                processing.</p>
+                            <div class="divider"></div>
+                            <h4 id="ROSEQuorumSensingModule" class="scrollspy">Quorum Sensing Module</h4>
+                            <p>Using community with different compartments would further improve the signal
+                                amplification, as known, quorum sensing is a system for stimulation(some signaling
+                                molecules called autoinducers) and response(receptor that will transcript certain genes)
+                                that is related to the bacterial concentration in the system. Many gene expression is
+                                naturally regulated by quorum sensing, which is also treated as social interaction in
+                                bacterial community.</p>
+                            <p>In some gram-negative bacteria, such as <em>Vibro fischeri</em>, biosynthesis of
+                                autoinducers is used for yield of bioluminescence through luciferase. The signaling
+                                molecule used by <em>V. fischeri</em> is an acylated homoserine lactone(AHL),called
+                                N-(3-oxohexanol)-homoserine lactone, which can be engineered into <em>E. coli</em>. The
+                                signaling molecule will be produced in the cytoplasm using LuxI synthase enzyme and
+                                then secreted through the cell membrane to the extracellular surroundings. When
+                                N-(3-oxohexanol)-homoserine lactone begins diffusing back into cells or other bacteria,
+                                LuxR will recognize the existence of N-(3-oxohexanol)-homoserine lactone with a
+                                threshold concentration, about 10ug/mL. Finally, target gene such as LuxlCDABE will be
+                                activated with LuxR and N-(3-oxohexanol)-homoserine lactone complex.</p>
+                            <p>It has been proved that LuxR has evolutionary conservation in many gram-negative
+                                bacteria, which enables <em>E. coli</em> to receive AHL from other bacteria. Thus we
+                                engineer Bacteria I to release quorum sensing molecules to Bacteria II for exponentially
+                                amplified results.</p>
+                            <p><img src="https://static.igem.org/mediawiki/2015/f/f3/RBS.png"/></p>
+                            <p>To achieve antibiotic concentration corresponding to AHL production, AHL produced by
+                                Bacteria I will be regulated by antibiotic substance sensing promoters. And in Bacteria
+                                II, LuxR will be directly expressed regulated by lac operon, triggered by IPTG. With
+                                treatment of IPTG, LuxR will be constitutively expressed to ensure LuxR-AHL complex will
+                                initiate the expression of required genes.</p>
                              </p>
-                             <br />
+                             <div class="divider"></div>
-                            <p class="divider"></p>
+                             <h4 id="ROSESecondLevelAmplificationModule" class="scrollspy">Second Level Amplification
-                             <h3 id="adhesion-module" class="scrollspy">Adhesion Module</h3>
+                                 Module</h4>
-                            <p><strong>What Material we Implement?-Theoratical Analysis</strong>
-                                 <br>In order to produce slight deformation of polymer membrane, we need to find a proper membrane to form this phenomenon. According to our modeling, see more in <a href="http://tower.im">Modeling-Determination of Polyer Membrane Candidate</a>, pressure caused by adhesive membrane is about 10^-4 Pa. Consequently, the wavelength of laser about 650nm is able to detect um-scale deformation and considering cm sqaured film, we need the Young's Modulus of membrane is smaller than 1GPa.
-                                <br><img src="image/QQ%E6%88%AA%E5%9B%BE20150810210009.png" alt="图片名称"></p>
-                            <p>The Young's Modulus of polyproylane is about 1.5Gpa, so it is selected as our candidate polymer membrane.</p>
-                            <p><strong>How to Achieve Stable Adhesion?</strong>
-                                <br>Bacterial adhereing to polymer membrane is one of the most important part in CACCI.In order to make bacteria binding tightly, chemically covalent bind of bacteria with polymer membrane maybe promising.</p>
-                            <p>So we decided to implement the parts from <em>2014 iGEM TU_Eindhoven</em>, and the whole system including these parts:</p>
-                            <ul>
-                                <li>
-                                    <p>DBCO-PEG4-NHS Ester (Dibenocyclooctyne-Polyethylene glycol-N-Hydroxysuccinimide), which covalently binding to polymer membrane.
-                                        <br><img src="image/1437880035x-954497725.png" alt="图片名称"></p>
-                                </li>
-                                <li>
-                                    <p>pAzF(p-Azido-L-Phenylalanine), a rare amino acid that would be implemented in bacterial transmembrane protein biosynthesis.
-                                        <br><img src="image/1437880130x-954497725.png" alt="图片名称"></p>
-                                </li>
-                                <li>
-                                    <p>In order to introduce pAzF into bacterial transmembrane protein, we need to mutate one of transmembrane protein, which is called COMPX. COMPX is able to form mutatedly with a special tRNA. The plasmid containing tRNA as following:
-                                        <br><img src="image/1436688284x-1376436670.png" alt="图片名称"></p>
-                                </li>
-                            </ul>
-                            <ul>
-                                <li>Covalent binding between DBCO and pAzF goes as following:
-                                    <br><img src="image/1436688789x-1376436664.png" alt=""></li>
-                            </ul>
-                            <p>To see more about constitution of our adhesion system, please refer to <a href="http://tower.im">Notebook-Protocol-Adhesion Preparation</a></p>
-                            <p><strong>Mutation of Transmembrane Protein OMPx in <em>E. coli</em></strong>
-                                <br>OMPx is a intergral outer membrane protein X from <em>Escherichia coli</em>, belonging to a family of highly conserved bacterial protein that promote bacterial adhesion to and entry into mamalian cells. Besides, these proteins as well play an important role in the resistance against attack by human complement system.
-                                <br> The modified COMPX structure is illustrated as below:
-                                <br><img src="image/1437880487x-954497725.png" alt="">
-                                <br> As planned, OMPx will be mutated at residue 16, from glycine to p-Azido-L-Phenylalanine, zooming in the mutated site in orange:
-                                <br><img src="image/1436688800x-1376436664.png" alt=""></p>
-                            <p><strong>The Covalent Reaction Model</strong>
-                                <br>Consequently, the model of adhesion goes like this: modifed bacterial containing special p-Azido-L-Phenylalanine transformed tRNA carry OMPx with mutated p-Azido-L-Phenylalanine site. The laminated side in pAzF will covalently bind to alkynyl in DBCO-PEG4-NHS ester, and consequently form chemical reaction.
-                                <br><img src="image/1437880652x-954497725.png" alt="图片名称"></p>
-                            <br />
-                            <p class="divider"></p>
-                            <br />
-                            <h3 id="optical-interference-module" class="scrollspy">Optical Interference Module</h3>
-                            <p><strong>What is Optical Interference?</strong>
-                                <br>Interference, in general physics, is a phenomenon when two waves superpose to form a resultant wave with a greater or lower amplitude, respectively called as constructive interference and destructive interference.
-                                <br><img src="image/QQ%E6%88%AA%E5%9B%BE20150810214624.png" alt="Mechiansm of Genereal Interference">
-                                <br>As for optical interference, because of high frequency of light waves(about 10^14 HZ), is it impossible to detect using current detector. Instead, only the intensity of an optical interference patteren can be observed, which is proportional to the square of the average amplitude of the wave.
-                                <br><img src="image/3800a8e0fb37906a8c6e138d105e2010.png" alt="图片名称">
-                                <br>The requirements of light source should be monochromatic, which means waves have a single frequency. Generally, a laser beam, in reality, approximates more closely to a monochromatic source and its straightforward generates interference
-                                <br><strong>Michelson interferometer</strong>
-                                <br><strong>Construction of NDM</strong></p>
-                            <br />
-                            <p class="divider"></p>
-                            <br />
-                            <h3 id="processing-module" class="scrollspy">Processing Module</h3>
-                            <p><strong>Working Curve Construction</strong>
-                                <br><strong>GUI</strong></p>
+                            <p>In addition of signal amplification through quorum sensing, an artificial genetically
+                                engineered cascade magnification circuit is also implemented in ROSE construction.</p>
+                            <p><img src="https://static.igem.org/mediawiki/2015/2/25/Cl.png"/></p>
+                            <p>In this cascade signal circuit, LuxR-AHL complex trigger the activation of promoter Lux,
+                                then express cI derived from λ phage. Since cI is a highly efficient repressor that is a
+                                low concentration of cI will completely repress the efficiency of λ promoter. As a result,
+                                EGFP will be drastically and negatively regulated when treated with antibiotic
+                                substances.</p>
+                            <p>It is notable that this second level amplification module won't directly expand the signal
+                                intensity, in another way, it will indirectly enlarge the resolution of different
+                                antibiotic concentration gradients. Our theoretical modeling also illustrate a higher
+                                resolution implementing this amplification circuit.</p>
+                            <div class="divider"></div>
+                            <h4 id="ROSEIntegrativeModificationonBacterialPermeability" class="scrollspy">Integrative
+                                Modification on Bacterial Permeability</h4>
+                            <p>Our ROSE construction is systematically not only include quorum sensing and logic
+                                amplification, but containing some permeability improvement by inhibition of efflux system
+                                and overexpression of exogenous porin. Nonpolar residues face outward so as to interact
+                                with the nonpolar lipid membrane, whereas the polar residues face inwards into the
+                                center of the beta barrel to interact with the aqueous channel.</p>
+                            <p><strong>Porin: OprF from <em>Pseudomonus aeruginosa</em></strong> Porins are beta barrel
+                                proteins crossing a celluar membrane and act as a pore through which molecules are able
+                                to diffuse.</p>
+                            <p>In <em>E. coli</em> different porins, for example OmpF and OmpC is expressed endogenously
+                                in different situations for different molecules. But these natural occurring porines are
+                                only permeable for molecules like sugars smaller than 600 Da. And more importantly,
+                                implement of micF, an antisense ncRNA may inhibit the material diffusion through OmpF.
+                                Therefore, it is necessary to import an exogenous porin with higher effiency of small
+                                molecular tranportation, such as OprF derived from <em>Pseudomonas aeruginosa</em>.</p>
+                            <p>OprF represents one of the largest pore sizes on bacterial outer membranes, allowing
+                                diffusion of polysaccharides in a range of 2000 to 3000 Da. The overexpression of OprF
+                                in <em>E. coli</em> will drastically improve the antibiotic absorption capability, like
+                                antibiotic condensation occured in <em>E. coli</em>.</p>
+                            <p><strong>Viroporin: SCVE from SARS Virus</strong> Viroporins are similar to porins in that
+                                they oligomerize within the membrane to form pores and this oligomerization often occurs
+                                between hydrophobic, transmembrane and alpha-helical domains of the proteins.</p>
+                            <p>SCVE, SARS Caronavirus Envolope protein, is a kind of effective viroporin within 76
+                                residues that is found normally pentamerized in bacteria. SCVE is permeable for many
+                                small molecules, which is originately designed for apoptosis of cells triggered by
+                                virus. Implementation of SCVE in ROSE may potentially improve the permatilibty of small
+                                molecules including antibiotics.</p>
+                            <p><strong>Inhibition of Efflux System using CRISPR</strong> Efflux system is a kind of
+                                mechanism found in microbes responsible for many moving molecules, especially some toxic
+                                substances, microbiotics.</p>
+                            <p>
+                                According to previous research, efflux system contributes a lot for bacterial antibiotic
+                                resistance, or multidrug resistance(MDR). For example, in <em>E. coli</em>, AcrAB efflux
+                                system has a physiological role of pumping out many bile acids, fatty acids to lower
+                                their intracelluar toxicty. It would be an attemptation to go directly reverse to
+                                antibiotic resistance, that is to inhibit the function of efflux system. In order to
+                                completely inhibit the major efflux system of <em>E. coli</em> in genetic level, CRISPR
+                                would be an impressive solution for totally deletion of the function of efflux system.
+                            </p>
+                            <br/>
+                            <p>
+                                Clutersted reularly interspacaed short palindromic repeats, abbreviated as
+                                <em>CRISPR</em>, are some fragements in prokayotic DNA with short repeats. Natually,
+                                CRISPR/Cas system is an important immune system that is able to resist the existence and
+                                reproduction of exogenous genetic materials, directly recognizing and then cut these
+                                genetic fragments, such as phages and plasmids, by CRISPR spacers, which is quite
+                                similar to RNA interference(RNAi) discovered in eukaryotic organisms. Cas9, which is
+                                CRISPR Associated Protein 9 in brief, is an RNA-guding DNA endonuclease enzyme
+                                associated with the CRISPR segments derived naturally from * Streptococcus pyogenes* and
+                                other bacteria. It has been showed that Cas9 plays an important role in memorizing,
+                                interrogating and ultimately cleaving these foreign DNA. Mechanism of cleavage is to
+                                unwind the foreign DNA and check whether there is a about 20 bp fragments that is
+                                complementary to guide RNA (gRNA, in brief). When the recognition result turns to yes,
+                                Cas9 will cleave the foreign DNA. Nowadays, with proper modification, CRISPR/Cas9 has
+                                become a common gene editing tool, being able to cut the target gene with gRNA design.
+                            </p>
+                            <p>
+                                When it comes to artificial design of CRISPR/Cas9 system, how to sucessfully achieve
+                                recruitment of Cas9 depends on the structure of gRNA. In general, gRNA consists of 2
+                                main parts, the former part is about 20 bp RNA sequences, which is exactly complementary
+                                to target genes, and it will be more impressive if these complementary sequence is at
+                                the beginning of the transcrption sites of gene. And the latter part is a secondary
+                                structure part which finally forms as hairpin used to recruit Cas9. During engineered
+                                section, this structual part can directly import from iGEM parts and what we need to
+                                take care is the fragment complementary to efflux system gene.
+                            </p>
+                            <p>CRISPR/Cas9 System for AcrB:</p>
+                            <p><img src="https://static.igem.org/mediawiki/2015/4/49/CRISPR_Cas9_System_for_AcrB.png"/></p>
+                            <p>CRISPR/Cas9 System for EmrE:</p>
+                            <p><img src="https://static.igem.org/mediawiki/2015/f/fc/CRISPR_Cas9_System_for_EmrE.png"/></p>
+                            <div class="divider"></div>
+                            <h4 id="ROSETheoreticalAdvantage" class="scrollspy">Theoretical Advantage</h4>
+                            <p><img src="https://static.igem.org/mediawiki/2015/1/1b/20150906Circuit.jpg"/></p>
+                            <div class="divider"></div>
+                            <h4 id="ROSEBibliography" class="scrollspy">Bibliography</h4>
+                            <p>
+.<i>A bacterial reporter panel for the detection and classification of antibiotic
+                                substances <b>Microbial Biotechnology (2012) 5(4), 536-548</b>Sahar Melamed, Shimshon
+                                Belkin</i>
+                            </p>
+                            <p>
+.<i>Signal-Amplifying Genetic Circuit Enables In Vivo Observation of WeakPromoter
+                                Activation in the Rhl Quorum Sensing System<b>Wiley InterScience 2005</b>DOI:
+.1002/bit.20371</i>
+                            </p>
+                            <p>
+.<i>MicF: An Antisense RNA Gene Involved in Response of Escherichia coli to Global
+                                Stress Factors <strong>2001 JMB</strong> doi:10.1006/jmbi.2001.5029</i>
+                            </p>
                          </div>
                      </div>
@@ Line 475: / Line 952: @@
                              <ul class="section table-of-contents">
                                  <li>
-                                     <a href="#who-is-cacci-">Who is CACCI?</a>
+                                     <a href="#ROSEwhatis">What is ROSE?</a>
                                  </li>
-                                <div class="divider"></div>
                                  <li>
-                                     <a href="#chemotaxis-modification-module">Chemotaxis Modification <br/>Module</a>
+                                     <a href="#ROSESystematicOverviewofROSE">Systematic Overview <br/>of ROSE</a>
                                  </li>
-                                <div class="divider"></div>
                                  <li>
-                                     <a href="#adhesion-module">Adhesion Module</a>
+                                     <a href="#ROSEAntibioticSubstanceSensingModule">Antibiotic Substance<br/> Sensing
+                                        Module</a>
                                  </li>
-                                <div class="divider"></div>
                                  <li>
-                                     <a href="#optical-interference-module">Optical Interference <br />Module</a>
+                                     <a href="#ROSEQuorumSensingModule">Quorum Sensing Module</a>
                                  </li>
-                                <div class="divider"></div>
                                  <li>
-                                     <a href="#processing-module">Processing Module</a>
+                                     <a href="#ROSESecondLevelAmplificationModule">Second Level<br/> Amplification Module</a>
+                                </li>
+                                <li>
+                                    <a href="#ROSEIntegrativeModificationonBacterialPermeability">Integrative
+                                        Modification <br/>on Bacterial Permeability</a>
+                                </li>
+                                <li>
+                                    <a href="#ROSETheoreticalAdvantage">Theoretical Advantage</a>
+                                </li>
+                                <li>
+                                    <a href="#ROSEBibliography">Bibliography</a>
                                  </li>
                              </ul>
@@ Line 499: / Line 983: @@
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