Team:Tokyo Tech/AHL Assay/AHL Reporter Assay

3-2-1 AHL Reporter Assay

1. Introduction

This section describes evaluation of the module of Quorum Sensing. Snow White is dead after eating a poisoned apple that is given by Queen. In order to trace this scene with E. coli, establishing cell-cell communication is necessary.

AHLs, which stand for [N-]acyl-homoserine lactones, are small signaling molecules and are employed in the bacterial “Quorum Sensing” system. In our project, we decided to use three AHLs studied well by several preceding iGEM Teams; the AHL molecules used here were C4HSL (hereafter referred to as C4), 3OC6HSL (C6), and 3OC12HSL (C12). Using these molecules, we enable the E. coli cells in a test tube to communicate and interact. By the experiments in this section, the module of Quorum Sensing was evaluated.

2. Summary of the Experiment

The purpose of the experiments in this section is to evaluate the activities of Prhl (BBa_I14017), Plux (BBa_R0062), and Plas (BBa_R0079) promoters upon addition of different AHLs. When C4, C6, and C12 internalize into E. coli cells, these molecules are accepted by the corresponding receptor proteins, RhlR, LuxR, and LasR, respectively. Then, the RhlR-C4, LuxR-C6, and LasR-C12 complexes activate/repress transcription from the Prhl, Plux, and Plas, respectively. However, it should be considered that illegitimate reactions of an AHL to non-native receptor proteins also occur to some extent. This phenomenon is called crosstalk. We wanted to seek the best combination of AHL systems for co-culture of Snow White coli and Queen coli. To this end, three plasmids shown below (Fig.3-2-1-5) were constructed and RFU of GFP/ Turbidity after adding each AHL to culture medium was measured.

3. Results

The experiments proved that our parts worked as expected (Fig.3-2-1-3-1).
The ratio of promoter strength was Prhl(BBa_I14017) : Plux(BBa_R0062) : Plas(BBa_R0079)= 2 : 40 :1.
The Lux system, which is usually activated by C6, reacted not only to C6 but also C12 (crosstalk). The reaction rate to C12 was half as that to C6. The Rhl system, which is usually activated by C4, reacted not only C4 but also C6 (crosstalk). The reaction rate to C6 was 0.78-fold relative to that to C4.


Fig. 3-2-1-3-1 RFU of GFP/ Turbidity of AHL Reporter Assay

4. Discussion

Considering different expression level from the AHL-inducible promoters shown above, we should find and choose the appropriate combination of the AHL molecules, the receptor proteins, and the AHL-inducible promoters for our final genetic circuits. The above results implied that, for expression induction from Plux(BBa_R0062), using C12 but not C6 was better to orchestrate the promoter activities. However, the promoter activity of Prhl(BBa_I14017) was still estimated to be incomparable to that of Plux(BBa_R0062). In fact, when our final circuits was simulated, it was found that the Prhl(BBa_I14017) activity was too weak compared to Plux(BBa_R0062) to operate our final circuits successfully (see the Modeling page). Taken together, we concluded that improvement of Prhl(BBa_R0071) was necessary to increase its promoter activity and orchestrate the promoter activities (see the Rhl System Assay page).

5. Materials and Methods

5-1. Construction

-Strain
All the plasmids were prepared in XL1-Blue strain.

-Plasmids
rbbs A. Pcon-rbs-rhlR(BBa_C0071)-tt-Prhl(BBa_I14017)-rbs-gfp (pSB6A1) (Fig. 3-2-1-5-1)



Fig. 3-2-1-5-1 Pcon-rbs-rhlR-tt-Prhl-rbs-gfp (pSB6A1)

B. Pcon-rbs-luxR(BBa_C0062)-tt-Plux(BBa_R0062)-rbs-gfp (pSB6A1) (Fig. 3-2-1-5-2)



Fig. 3-2-1-5-2 Pcon-rbs-rhlR-tt-Prhl-rbs-gfp (pSB6A1)

C. Pcon-rbs-lasR(BBa_C0179)-tt-Plas(BBa_R0079)-rbs-gfp (pBS6A1) (Fig. 3-2-1-5-3)



Fig. 3-2-1-5-3 Pcon-rbs-lasR-tt-Plas-rbs-gfp (pBS6A1)

D. Pcon-rbs-gfp (pSB6A1) …Positive control (Fig. 3-2-1-5-4)



Fig. 3-2-1-5-4 Pcon-rbs-gfp (pSB6A1)

E. pSB6A1 …Nagative control (Fig. 3-2-1-5-5)



Fig. 3-2-1-5-5 pSB6A1

-Medium
LB medium A
LB medium containing ampicillin (50 microg/ mL)





5-2. Assay Protocol

The following experiments are performed at 37℃ unless otherwise stated.

1) Prepare overnight cultures for each sample in 3 mL LB medium A with vigorous shaking.

2) Dilute the overnight cultures to 1 / 60 in fresh LB medium A (1.2 mL).

3) Incubate the fresh cultures for 1 h with vigorous shaking.

4) Add 500 microM C4, 500 microM C6, 500 microM C12 or DMSO to each 500 microL sample at the final concentration 10 microM.

5) Incubate the samples for 4 h with vigorous shaking.

6) Add 100 microL of the samples into each well of a plate reader.

7) Measure RFU of GFP at 490 nm as an exciting wavelength, 525 nm as an emission wavelength.

8) Measure the turbidity at 600 nm.

6. Reference


(1) Kendall M. Gray et al. (1994) Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. Journal of Bacteriology 176(10): 3076–3080