Difference between revisions of "Team:USTC/Description"

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.

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:

1. Chemotaxis Modification: to increase the bacterial mobility, modification based on E. coli chemotaxis related to the parts cheZ will be introduced into bacteria.
2. 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.
3. 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.

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.

  

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

  

• 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:

  

• Covalent binding between DBCO and pAzF goes as following:

  

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.

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

1. Comparative studies of bacteria with an atomic force microscopy operating in different modes. Ultramicroscopy 86 (2001) 121–128 A.V. Bolshakova, I.V. Yaminsky.

2. 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.

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.

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.

This is our design of hardware part.

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.

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.

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".

Then let's see the screen , keyboard and mouse.

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.

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.

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.

Software and Fringe analysis

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

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

Methods

(1)

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

(2)

1. Take a series of photos at the same position in a short time.
2. Superpose these photos to sharp the edge of every object.
3. Calculate the number of fringes on vertical direction(y axis).
4. 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

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

Code

Demo

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.

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

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.

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.

• 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