final posterConclusion
Nowadays, the emerging issue in bacterial infection is the appearance of multiple drug-resistant (MDR) bacteria and extensive drug-resistant (XDR) bacteria. Yet the development of resistant strains of bacteria could be limited by the judicious use of antibiotics. Knowing the antibiotic pattern of the invading bacteria is of importance in clinical treatment. Among nosocomial infection, urinary tract infections, pneumopathy, and infections of surgery site are most common because of the formation of biofilm on the surface of catheter, endo-tube. Therefore, identification of bacteria and new drugs are required.
QUORUM SENSING ARRAY NATIONAL TAIWAN UNIVERSITY
To date, it has been found that Pseudomonas aeruginosa has at least four quorum sensing systems: Las system, Rhl system, Qsc system, and PQS system. Las, Rhl and Qsc system all contain an AHL- activated receptor. Las system and Qsc system use 12-C AHL as Quorum Sensing(QS) molecules; on the other hand, Rhl system prefers to use 4-C AHL as QS molecules. However, interestingly, PQS system is not activated through AHL, but a quinoline derivative: 2-hep- tyl-3,4-dihydroxyquinoline. Once PQS binds to pqsR, pqsR, it can further activates two operons: pqsABCDE and phnAB. These two operons encode the enzymes that can synthesize PQS, and pqsE can catalyze PQS to become phenazine, which is related to virulence factors.
PQS System
Background
RESULT
MODELING
Conclusion
Our IGEM project aims to tailor an instant bacteria identification array using quorum sensing molecules. We proposed a novel method to identify bacteria by the expression pattern of many QS receptors from the intensity of fluorescent signals. We designed both positive feedback and negative regulation circuits insides plasmids containing quorum sensing receptors conjugated with florescent proteins. Different receptors target different kinds of AHL molecules depending on its carbon number of the acyl group. In addition to AHL, we also developed novel biosensor—PQS for new type of quorum sensing molecule in igem—quinolones. Our functional testing includes using ELISA plate reader and flow cytometer to calibrate the standard diagram of different biosensor. Finally, the supernatant of bacteria were taken into ex- periments. We hope by collecting enough data of different clinical bacterial strains we are able to build up a new system for bacteria identification.
N U Taida
Human Practice
We have arranged a series activities to both meet the actual needs for nosocomial infection detection as well as to promote the ideas of synthetic biology and iGEM. We had visited the Department of Laboratory Medicine in NTUH ; we also developed a workshop for high school students. We cooperated with some student clubs and other iGEM teams in Taiwan, including holding NTU-Taida x Design Thinking workshop, joining in the International iGEM Conference held in NCTU and helping in some parts of each other’s projects. We had funded a club called, “Investigator x iGEM Taiwan” to hold these kinds of events regularly!
Gram-positive bacteria generally use auto-induc- ing peptides as quorum sensing molecules. By binding to its cognate membrane-bound sensor kinase or intracellular receptor, the quorum sens- ing signal is transmitted back and spread to other bacterial cells, creating an inter-cellular communi- cation network. Clinically important Gram-positive bacteria in- clude Staphylococcus aureus and Streptococcus pneumoniae.
Due to gram-negative bacilli membrane structure, quorum sensing molecules diffuse through the cell membrane and bind with intracellular receptors. Generally speaking, quorum sensing system in gram negative bacteria functions as follows (Take LasI/LasR system for example): The LasI gene encodes an autoinducer syn- thase(LasI), and this autoinducer synthase produc- es quorum sensing molecules called acyl-homoser- ine lactone(AHLs). Another gene LasR encodes for the response regulator of the AHLs. Regula- tors bind with autoinducers and form complexes. They bind on target promoters, and then either ac- tivate or inhibit relevant down-stream genes.
Gram positive Gram Negative
FUTURE PLAN (1) More quorum sensing sensors should be constructed in order to expand its bacteria sensing spectrum in hope of building a quorum sensing based array. (2) More clinical species should be tested by these receptors, and its application should be promoted to clinical patient samples. (3) Recent studies of drugs interfering quorum sensing mole- cules have given quorum sensing array a possibility of support- ing diagnosis and prescription.
According to our results, we believed that we have built the prototype of a quorum sensing array. There are several steps that must be reached before this biosensor can be put into practical use :
We hope that someday, quorum sensing signals may not only be used to differentiate bacterial strains but support doctors to prescribe drugs.
(1) Apart from the traditional AHL-based quorum sensing molecules, PQS uses quinolone as its signal molecule. We cloned original biobrick PQS receptor and its promoter sequence. (2)We constructed three types of quorum sensing biosensor circuits, and tested its function of dosage response towards time. (3) Combined 4 types of quorum sensing receptors and proposed a prototype of quorum sensing array. (4) We tested clinical bacterial sample when placed with our biosensors and proved that it is able to identify different strains of bacteria and it is possible to be used clinically. (5) We compared the ELISA plate reader results and flow cytometry results and confirmed that single cell fluorescence expression fits the results of single cell modeling. ELISA plate reader fits the re sults of 2D model.
Single Cell modeling Each cell can detect AHL and generate GFP with different behaviors. We run 1000 times for 3 initial conditions (AHL conc = 10-3, 10-5, 10-7) Distribution of GFP concentration from simulation can be compared with reality.
2D diffusible model We use pixels to construct 2D space, each pixels can be configured with specific parameters to described its behavior. For example, it can set all reaction rate constant to 0 to simulate the extracellular environment. It can set the diffusion constant of proteins to 0 to simulate the behavior of cell membrane
Compare simulation model with real data -Single cell reach max GFP intensity or con- centration within 1 hours -2D celldivision model can fit well with real data in microplate readerwith R2 > 0.96
A. Rhl-mCherry normal circuit B. Rhl-GFP with positive feedback C. Rhl-mCherry with CI-pCI circuit
We used several kinds of modeling to simulate the fluorescence expression of our biosensors.
In order to understand the “actual” bacterial fluores- cence expression during each time point, we use flow cytommetry to detect individual fluorescence. This is the flow cytometry results of BBa_K1157006 under different time and concentration.
(3)Flow cytommetry results
B. Las quorum sensing |BBa_K575024
C. Lux quorum sensing |BBa_T9002
D. PQS quorum sensing |BBa_ K1157017 & |BBa_ K1157019
Fundamental circuit
Circuit Design We have designed two kinds of circuits, based on the ways mentioned early. One has a positive-feed- back loop, while the other works on a negative-regulation mechanism. We also made basic circuits without any feedback, as the comparison of our designs.
It was said by team Northwestern, 2011 that if the reporter region was put inferior in the circuit, the response would not be obvious enough to detect. Due to curiosity, most of our circuits were made in two forms, with the reporter putting either forward or afterward, in order to reproduce the phenome- non and try to explain it.
The basic design is a double level devise. It contains a con- stitutive expression quorum sensing receptor, and a reporter regulated by QS regulated pro- moter.
Based on the fundamental one, we involved a positive-feedback loop in this time. A QS receptor gene was added in between the QS promoter and the reporter.
Referring to a paper in 2005, the negative regulation design in- volves CI gene in the circuit, using a pre-produce mechanism rather than post-produce to reduce reac- tion time. And still, the positive feedback of QS receptor is re- served in this circuit.
Positive feedback Negative regulation
We used ELISA plate reader to test fluorescent expression under different concentration of AHLs. Data are recorded every hour for 4-6 hours.
Quantitative experiments
After using AHL molecules as our testing targets, we use clinical species as our functional testing targets. We use the bacterial supernatant as our testing sample. We use ELISA plate reader to read the fluorescence after 4 hours of reaction of the supernatant and biosensor.
(2) Cinical bacterial species testing
(1) AHL dosage effect and fluorescence