TraI Assay

Introduction

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Quorum Sensing is cell-to-cell communication system used by variety of microorganism. Signal molecular used in Quorum sensing has variety of chemical structure. LuxI is synthesis gene of 3OC6HSL and TraI is synthesis gene of 3OC8AHL. Chemical structures of these molecules are shown in Fig. 1.

LuxR gene express intracellular LuxR receptor. Signal molecular and this receptor form complex. This complex interacts with responsive promoters, Plux and regulates transcription of downstream genes. The concentration of signal molecular increase with cell density. By using this system, microorganism assess their local density and regulates gene expression.
In previous study, a novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription factor TraR was developed (1). In this system, expression of downstream genes of CMV minimal promoter is induced in the presence of signal molecular 3OC8HSL. Therefore, we chose 3OC8HSL as a signal molecule and tried to make E.coli to produce 3OC8HSL.

Summary of experiment

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In this section, we confirmed whether E. coli expressing Tral protein truly produced signal molecules, AHL (Acyl Homoserine Lactone).
To achieve this goal, we constructed two types of E. coli. One is the “Sender” E. coli which produces AHL and the other is the “Reporter” E. coli which expresses GFP in the presence of AHL.
To begin with, we evaluated whether “Reporter” cell could express GFP dependent on AHL by culturing them in liquid LB medium containing various concentrations of AHL (0.1nM-1000nM).
In previous study, value of RFU for AHL concentration is known to follow Hill's equation(2). We obtained the parameters of Hill's equation from the data and calculated the concentration from the value of RFU.
Then we confirmed whether the “Sender” could produce AHL. The supernatant of the “Sender” cell’s medium was added into the medium of “Reporter” cells and the production of AHL was confirmed by the expression of GFP from the “Reporter” cells.

Following plasmids were introduced into E. coli.
Reporter E.coli
By introducing plasmids shown in Figure. 2, E.coli will be able to produce gfp in response to 3OC8AHL signal and 3OC6AHL signal.

Sender E.coli
We create Sender E.coli by introducing a plasmid shown in Fig. 3.
Sender E.coli constantly produce signal molecule, 3OC8AHL because TraI gene is placed at downstream of constitutive promoter, Ptet.

Results

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Reagent Assay
LuxR protein is a receptor for 3OC6HSL signals. However, previous study showed that it can also bind to other kinds of AHL, such as 3OC10HSL(3). We confirmed that LuxR could also respond to C8 signals as sensitive as respoding of 3OC6HSL signals. Receiver E.coli’s RFU (Reletive Fluoroscent Units) in each AHL concentration (0.01 nM ? 1000 nM) is shown in Fig. 4. Detection limit was over 10nM in case of 3OC6HSL and 3OC8HSL. RFU values were almost same over 100nM.

Error bar have a same width as standard deviation (n=3).

Based on the data which is shown in Fig. 4, parameter was obtained to fit Hill’s equation.
Hill’s equation is shown in Eq. 1

The values of Parameter is shown in Table. 1
Parameter “a” represents leakiness of Receiver E. coli’s gfp. Even in the absence of AHL of Signal molecule, it is known that no downstream gene below Plux is not transcribed at all and gfp "leaks" somewhat. “b” is the value of RFU when AHL binds to all receptor and is completely induced. “n“ is a value called Hill coefficient, and when this value is 1 or more, it is said that there are multiple binding sites. “Km” is the AHL concentration when a ligand (AHL) binds to half of the receptor, and this value represent the detection sensitivity of the reporter E.coli. It was found that both AHLs can be detected with sensitivity of tens of nM.

Actual measurement value and Theoretical formula is shown in Fig. 5.
In both graphs, the RFU value increased greatly from 10 nM to 100 nM, and it was found that more than 100nM of AHL, can not be directly quantified.

Supernatant Assay
Temperature dependence of AHL production.
We found that Amount of C8 production is depend on temperature. RFU was 14 folds larger than DH5α.
3OC8HSL concentration of TraI culture in37℃ was nM.　The RFU values of mixture in which Sender E.coli was cultivated in25℃ exceeded the detection limit.

Discussion

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We confirmed that E. coli can produce over 200nM of 3OC8AHL. However previous study(1) showed that 20μM of C8 is required to activate the target gene in human cells. Therefore, we need further improvement of C8 production.
Result of Figure.5 shows temperature dependence of 3OC8AHL production. TraI is derived from soil microorganism A. Tumefaciens. It is rarely happen that Temperature of the soil rise above 37 ℃. Therefore it is considered that TraI protein does not work properly above 37℃.

Material and Methods

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Reagent assay
1. Cultivate Receiver E.coli in LB medium containing antibiotics for about 15hours
2. Dilute the culture to 1/200 with flesh LB medium containing antibiotics
3. Incubate the flesh culture for 2 hours
4. Mix 495μL of the culture with 5μL of DMSO solution (each DMSO is containing 100 microM,10microM...of AHL to reach final concentration 1microM 100nM...) in micro tube
5. Incubate the micro tube for 5 hours with Small shaking incubator in 37℃
6. Take 100μL of culture and Measure fluorescent (excitation wave length is 495nm, Measurement wavelength is 520nm) and absorbance (Measurement wavelength is 600nm)　Supernatant assay
Supernatant Assay
1. Cultivate Sender E.coli in LB medium for about 15hours
2. Centrifuge the culture 16,000rpm and 5minutes
3. Follow Reagent assay process (1~4) and Prepare Reporter culture.
4. Mix 250μL of sender culture’s supernatant with Reporter culture in micro tube.
5. Incubate the micro tube for 5 hours with Small shaking incubator in 37℃
6. Take 100μL of culture and Measure fluorescent (excitation wave length is 495nm, Measurement wavelength is 520nm gain is 45) and absorbance (Measurement wavelength is 600nm)

Appendix: Material and Method

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Reference

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(1). Neddermann P1, Gargioli C, Muraglia E, Sambucini S, Bonelli F, De Francesco R, Cortese R (2003) A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription factor TraR. EMBO Rep. 2003 Feb;4(2):159-65.
(2). https://2014.igem.org/Team:ETH_Zurich/modeling/qs
(3). https://2016.igem.org/Team:Tokyo_Tech/AHL_Assay/AHL_Reporter_Assay