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Experiment
Prhl reporter assay |
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1. Summary of the experiment |
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Prhl promoter is a regulatory part activated by RhlR in the presence of N-butyryl-homoserine lactone (also known as C4HSL). Existing Prhl promoter (BBa_R0071) has a low expression level even when it is activated. |
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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 (Prhl(RR): BBa_K1529320). |
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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) promoter showed about 20-fold higher in the fluorescence than that of the original Prhl promoter (BBa_R0071) (See 2. Results). |
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However, our Prhl(RR) promoter showed a significant leak in the absence of C4HSL(See 2. 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) promoter to LuxR binding site (Prhl(LR)) or the latter RhlR binding site to Lux binding site (Prhl(RL)). |
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Then, we inserted these promoters to the upstream of GFP coding sequence and measured the fluorescence intensity. Prhl(LR) promoter showed a higher maximum expression level, but also showed a significant leak like Prhl(RR) promoter. On the other hand, Prhl(RL) promoter had less leak while keeping the high expression level (See 2. Results). |
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Fig. 3-4-1. The design of our Rhl promoters |
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2. Results |
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We measured the 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) . |
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Fig. 3-4-2. The four promoters we tested |
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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 the promoters: Prhl, Prhl(RR), and Prhl(RL). |
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As Fig. 3-4-4 shows, when induced by C4HSL, Prhl(RR) promoter showed higher maximum expression level and higher leak than the original Prhl promoter. |
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Although Prhl(RL) promoter had lower maximum expression level compared to Prhl(RR) promoter, it had the highest induced/not-induced ratio. This means Prhl(RL) promoter has little leak. Therefore, we can say that Prhl(RL) promoter is the best improved Prhl promoter due to the advantages of less leak and higher expression level. |
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Fig. 3-4-3. The Fluorescence intensity of the cells (with positive and negative controls) |
Fig. 3-4-4. The fluorescence intensity of the cells with the original Prhl (BBa_R0071), Prhl(RR) (BBa_K1529320), and Prhl(RL) (BBa_K1529300)
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3. Materials and methods |
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| 3-1. Construction | |
| -Strain | |
All the samples were JM2.300 strain |
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| -Plasmids | |
A. Ptet-RhlR (pSB6A1), Prhl-GFP (pSB3K3) |
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Fig. 3-4-5. |
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B. Ptet-RhlR (pSB6A1) Prhl(RR)-GFP (pSB3K3) |
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Fig. 3-4-6. |
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C. Ptet-RhlR (pSB6A1) Prhl(LR)GFP (pSB3K3) |
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Fig. 3-4-7. |
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D. Ptet-RhlR (pSB6A1) Prhl(RL)-GFP (pSB3K3) |
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Fig. 3-4-8. |
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E. Ptet-RhlR (pSB6A1) PlacUV5-GFP (pSB3K3) ...Positive control |
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Fig. 3-4-9. |
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F. Ptet-RhlR (pSB6A1) promoter less-GFP(pSB3K3) ...Negative control |
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Fig. 3-4-10. |
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| 3-2. Assay Protocol | |
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. |
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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. |
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3. Incubate the fresh cultures in 37°C until the observed OD590 reaches 0.3 (Actual value 0.42). |
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4. Add 30 microL of 500 microM C4HSL or DMSO as listed below: |
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A-5 μM: A + C4HSL |
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A-0 μM: A + DMSO |
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B-5 μM: B + C4HSL |
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B-0 μM: B + DMSO |
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C-5 μM: C + C4HSL |
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C-0 μM: C + DMSO |
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D-5 μM: D + C4HSL |
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D-0 μM: D + DMSO |
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E-5 μM: E + C4HSL |
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E-0 μM: E + DMSO |
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F-5 μM: F + C4HSL |
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F-0 μM: F + DMSO |
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5. Incubate the samples at 37°C for 4 h. |
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6. Start preparing the flow cytometer 1 h before the end of incubation. |
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7. Take 200 microL of the sample, and centrifuge at 9000 Xg, 1 min, 4°C. |
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8. Remove the supernatant by using P1000 pipette. |
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9. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. |
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10. Dispense all of each suspension into a disposable tube through a cell strainer. |
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11. Measure fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company). |
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4. References |
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[1] “Simpson’s Paradox in a Synthetic Microbial System” John S. Chuang, Olivier Rivoire, Stanislas Leibler 9 JANUARY 2009 VOL 323 SCIENCE |
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