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 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.

Antibiotic substances containing several mechanism, rendering them to kill and inhibit bactieral 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 catogorization based on chemical structure including:

• Beta-lactam
• Aminoglycoside
• Amide alcohol
• Macrolide
• Polypetide
• Tetracycline
• Sulfonamides
• Quinolones
• Polymyxin
• Trimethprim
• Nitrofuran etc.

Ordered formal course of treatment, these antibiotic would effectly 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 familiy of the probiotics, and this has been associated with chronic diseases.

The prodcution of antibiotics in pharmceutical companies explosively expanded these years. Articifical 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 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.

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 Klebsiella pneumoniae from a Swedish patient of Indian origin in 2008 once may contribute to severe disaster on that broad of beta-lactan 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 emittes 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 scienctists 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 dificiencies, including:

• Not portable, many big and automatic equipments are indispensable for concentration detection.
• 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.
• Recent technology is still not sensitive enough and sometimes time consuming.

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

What is ROSE?

Reporter Operater with Sensing Engine, in brief-ROSE, 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.

Systematic Overview of ROSE

In general, ROSE as a community, containing two kinds of bactiera, among which Bactiera I specfically 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 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.

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.

Antibiotic Substance Sensing Module

Previous research from Sahar Melamed and Shimshon Belkin has already selected dozens of promoters responding to antibiotic concentration specificallly 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 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.

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 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.

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 exponentailly 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 consitutively expressed to ensure LuxR-AHL complex will intiate 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 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.

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.

Integrative Modification on Bacterial Permeability

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.

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 CRIPSR 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, CRIPSR would be an impressive solution for totally deletion of the function of efflux system.

Clutersted reularly interspacaed short palindromic repeats, abbreviated as CRIPSR, 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.

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

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. 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 interation using polylysine(PLL) and covalent interation by modifing 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 interation 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. Interference: Once the bacteria are adhered to the film and treated in water sample, their movement will bring about interference. The interference patterns will be different at distinct chemical molecule concentration.
4. Processing: These patterns will be captured by our CCD camera. Our control system containing interference pattern recognition will process these patterns, and finally a work curve will be established where the antibiotic concentrations and different interference patterns correspond. For friendly operation, graphic user interface (GUI) is established better for user utilization on this program.

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 demonstrate 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:

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 promosing 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 expriment 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 intergral outer membrane protein X from Escherichia coli, 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.

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.

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.