Team:Tokyo Tech/Experiment/Prhl reporter assay

1. Summary of the experiment

Rhl promoter (Prhl) is a regulatory part activated by RhlR in the presence of N-butyryl-homoserine lactone (also known as C4-HSL). Existing Rhl promoter (BBa_R0071) has a low expression level even when it is activated.

In order to improve this expression level, we designed a new Lux promoter which has two RhlR binding sites instead of two LuxR binding sites (PPrhl_RR: BBa_K1529320).

To evaluate the function of this promoter, we constructed Prhl_RR-GFP plasmids and measured the fluorescence intensity by flow cytometer. In the measurement, we confirmed that GFP under the control of Prhl_RR showed about ???-folds higher in the fluorescence than that of the original Prhl (BBa_R0071) (See results).

However, our Prhl_RR showed significant leak in the absence of C4HSL(See results). In order to lessen the leak and increase the maximum expression level, we newly designed two promoters, Prhl_LR (BBa_K1529310) and Prhl_RL (BBa_K1529300). These promoters have one LuxR binding site and one RhlR binding site. We changed either the upper RhlR binding site of Prhl_RR to Lux binding site (Prhl_LR) or the latter RhlR binding site to Lux binding site (Prhl_RL).

Then, we inserted these promoters to the upstream of GFP coding sequence and measured the fluorescence intensity. Prhl_LR showed higher maximum expression level, but also showed significant leak like Prhl_RR. On the other hand, Prhl_RL had less leak while keeping the high expression level (See results).

2. Results

We measured GFP expression with the four different promoters (Prhl (BBa_R0071), Prhl_RR (BBa_K1529320), Prhl_LR (BBa_K1529310) and Prhl_RL (BBa_K1529300)) by flow cytometer. Each promoter was tested in the presence and also in the absence of C4HSL (See Materials and Methods for detailed procedures).

Fig. 3-4-3 shows the fluorescence intensity detected by flow cytometer. Fig. 3-4-4 is the extracted data which shows the comparison of Prhl, Prhl_RR, and Prhl_RL

As Fig 3-4-4 shows, when C4HSL is induced, Prhl_RR showed higher maximum expression level and higher leak than the original Prhl.

3. Materials and methods

All the samples were JM2.300 strain

A. Ptet-RhlR (pSB6A1), Prhl-GFP (pSB3K3)

B. Ptet-RhlR (pSB6A1)　Prhl_RR-GFP (pSB3K3)

C. Ptet-RhlR (pSB6A1)　Prhl_LR-GFP (pSB3K3)

D. Ptet-RhlR (pSB6A1)　Prhl_RL-GFP (pSB3K3)

E. Ptet-RhlR (pSB6A1)　PlacUV5-GFP (pSB3K3) ...Positive control

F. Ptet-RhlR (pSB6A1)　promoter less-GFP(pSB3K3) ...Negative control

1. Prepare 2 overnight cultures for each samples A~F in 3 mL LB medium, containing ampicillin (50 microg /mL) and kanamycin (30 microg / mL) at 37°C for 12 h.

2. Dilute the overnight cultures to 1 / 100 in fresh LB medium (3 mL) containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) (→fresh culture). Make glycerol stocks from the remainders.

3. Incubate the fresh cultures in 37°C until the observed OD590 reaches 0.3 (Actual value 0.42).

4. Add 30 microL of 500 microM C4HSL or DMSO as listed below:

A-5 μM: A + C4HSL

A-0 μM: A + DMSO

B-5 μM: B + C4HSL

B-0 μM: B + DMSO

C-5 μM: C + C4HSL

C-0 μM: C + DMSO

D-5 μM: D + C4HSL

D-0 μM: D + DMSO

E-5 μM: E + C4HSL

E-0 μM: E + DMSO

F-5 μM: F + C4HSL

F-0 μM: F + DMSO

5. Incubate the samples at 37°C for 4 h.

6. Start preparing the flow cytometer 1 h before the end of incubation.

7. Take 200 microL of the sample, and centrifuge at 9000 Xg, 1 min, 4°C.

8. Remove the supernatant by using P1000 pipette.

9. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.

10. Dispense all of each suspension into a disposable tube through a cell strainer.

11. Measure fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).

4. References

[1] “Simpson’s Paradox in a Synthetic Microbial System” John S. Chuang, Olivier Rivoire, Stanislas Leibler 9 JANUARY 2009 VOL 323 SCIENCE