Difference between revisions of "Team:elan vital korea/Protocol"

4.
Other materials such as yeast and NaCl are stored and maintained under the responsibility of Gachon Molecular Biology Lab.
5.
We have to record the history of each material, including if plasmids/reporter cell/ test cell/ AHL have been frozen and if so, when it is used.

LB Agar Plates and Addition of Antibiotics

1.
We have used LB (solidified lysogeny broth), rich growth medium for E.coli, in our experiments almost all the time.

2.
All the needed chemicals including yeast extract must be prepared beforehand.

3.
Autoclave must be available.

4.
Growth on LB Agar plates provides colonies originating from one single bacteria cell.

5.
Just before pouring the solution into petri dishes, an antibiotic can be added for resistance selection.
We followed the normal working concentrations such as:
- chloramphenicol: 25 μg/mL
(Chloramphenicol stock is dissolved in ethanol)
In case of using ampicillin: 100 μg/mL
- normal stock concentrations:1000-fold

6. Material to make LB plates:
NaCl (Sodium Chloride)
Bacto™ tryptone
yeast extract
Bacto™ agar
ddH2O (triple distilled water)
1000x chloramphenicol or ampicillin

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7. Process
We usually make 1liter bottle for LB Agar
1) 200 mL LB prepared fresh, non-autoclaved
2) 3 g agar
3) Shake until all solids are dissolved
4) Autoclave for 20 min within 2 hr
5) Keep it cool until it reaches around 40-50 °C
6) Add 200 μL of 1000x chloramphenicol and gently stir it. Be careful not to shake the bottle too long/hard so that bubbles are created.
7) Pour into empty petri dishes just enough to cover the surface (~20 mL per plate). In case that bubbles are in the plate, heat the plate surface carefully with a burner only until the bubbles are burst but the solution is heated.
8) Leave the plates at room temperature around one hour until it is solidified.
9) Solidified plates should be turned upside down for a few hours at room temperature, then stored at 4°C.

LB Medium

CDC dramatically innovated the detection process by adopting the Advanced Molecular Detection (AMD), which combines the latest pathogen
identification technologies with bioinformatics and advanced epidemiology to more effectively understand, prevent and control infectious
diseases. Using those technologies, it is possible to rapidly look for a microbe's match among thousands of reference
samples in the microbe library. If no match is found, the whole genomic sequence
of the microbe's DNA code can be taken, then quickly analyzed using disease detective works and bioinformatics
to answer critical disease-response questions. However, this new method, while it sounds very interesting, is not to be
completed until 2020, and still requires incubation, as well as being expensive.

Our Hypothesis: Possibility of Using Quorum
Sensing for Early Detection

Our team, Elan Vital Korea, addressed the problem of rapidly detecting antibiotic-resistant bacteria. We were interested in
a rapid and efficient method of antibiotic resistance detection, and we believed that such a method could be engineered
using quorum sensing. Our hypothesis was that we would be able to use quorum sensing – a method bacteria
use to communicate with each other – to make the cells quickly report the existence of antibiotic-resistant bacteria

By quorum sensing, bacteria can perform many cooperative functions, such as biofilm formation, antibiotic production, motility,
swarming, virulence, and much more. While most quorum sensing takes place between bacteria of the same species, there are
cases of interspecies quorum sensing. Auto-inducers affect the gene expression of the bacteria once they reach
a certain concentration threshold. Bacteria using quorum sensing usually produce small amounts of
auto-inducers, so that the concentration of auto-inducers are affected by the concentration of the bacteria.
In other words, quorum sensing, in essence, regulates gene expression in response to cell density.
Using quorum sensing, bacteria are able to act in unison, as if they were a single organism.

Quorum sensing is widely used by various bacteria for various functions, so each uses a slightly different auto-inducer
so the signals are not mixed up. In general, gram-negative bacteria use a class of molecules called N-acyl
homoserine lactones, or AHL, while gram-positive bacteria use short processed polypeptides.

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For example, the picture below represents the quorum sensing mechanism in the bacteria vibrio fisheri. Vibrio fisheri is a bacteria
that produces bioluminescence, and is famous for revealing quorum sensing for the first time. Vibrio fisheri uses quorum sensing
to produce light in high cell density, and researchers first discovered quorum sensing from examining vibrio fisheri.

In vibrio fishri, quorum sensing involves LuxI and LuxR as well as AHL. LuxI is the protein that produces AHL, and LuxR forms a complex
with AHL to affect the regulation of genes. In this case, it produces luciferase, which produces bioluminescence. Furthermore, the process
also boosts the production of LuxI, which creates a positive feedback loop. This AHL-LuxR quorum sensing mechanism is one of the most well
known gram-negative quorum sensing pathways, and it can be
engineered to affect almost any coding sequence we like.

For the project, we have developed a reporter cell that expresses GFP in the presence of the QS signaling molecule acyl homoserine
lactone (AHL). Our test cells (which act as a simulation of antibiotic-resistant bacteria) express lactonase,
which breaks down AHL. In our experimental system, test cells should signify their presence by breaking down AHL and
preventing GFP expression in reporter cells.

Experiment: Process and Results

There are many ways of utilizing quorum sensing for medicinal use, and one of the most intuitive and most well-known methods is quorum quenching.

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Quorum quenching takes advantage of the fact that quorum sensing also plays a role in expressing virulence, and interferes with the quorum sensing that
produces virulence. There are many ways of utilizing quorum sensing for medicinal use, and one of the most intuitive and most well-known methods is quorum quenching.
Quorum quenching takes advantage of the fact that quorum sensing also plays a role in expressing virulence, and interferes with
the quorum sensing that produces virulence. However, for our project this year, we decided to focus on engineering a detection method for antibiotic resistance.
For the project, we created a test plasmid and a reporter plasmid. We then transformed competent E. coli with the plasmids to produce a
test cell and a reporter cell. As shown in the picture below, the test cell produces lactonase, which breaks down AHL, a common auto-inducer in
gram-negative bacteria. And the reporter cell produces GFP (or luciferase) which creates a visible difference that we can detect.
Both plasmids were engineered using the BioBrick DNA recombination process. With such a set up, it will be possible to detect the presence of the test cell, or lactonase.

For the confirmation of our hypothesis, we conducted some experiments. Ideally, mixing AHL with the test cell will break down the AHL. And, adding
the reporter after that will not result in any fluorescence. But, if we do the same process with the control bacteria instead of the test cell,
there will be fluorescence. As theorized, the control experiments produced fluorescence, but the experiments with the test cell produced no fluorescence.

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