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