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Experiment
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1. Summary of the experiment |
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We added three improved C4HSL-dependent promoters with high maximum expression level by combinations of regulatory-protein binding sites. (Fig. 3-2-1-1). |
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First, we designed a improved Lux promoter which has two RhlR binding sites instead of two LuxR binding sites (Prhl(RR): BBa_K1529320) , as tried in a previous paper (Chuang 2009). To evaluate the function of this promoter, we constructed Prhl(RR)-GFP (BBa_K1529321) 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). |
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However, Prhl(RR) promoter showed a significant leak in the absence of C4HSL. High level of leakage is not suitable for the Company-Customer relationship because their mutualistic will be broke. |
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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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One of our new promoter, Prhl(RL) improved in its expression level while keeping the low leakage. GFP under the control of Prhl(RL) promoter showed about 7-fold higher in the fluorescence than that of the original Prhl promoter. The leak was no more than 2-fold high. |
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Although the other Prhl(LR) promoter showed a higher maximum expression level, it showed a significant leak like Prhl(RR) promoter. GFP under the control of Prhl(LR) promoter showed about 7-fold higher in the fluorescence than that of the original Prhl promoter. However, the leak showed no less than 25-fold high. Thus we used our improved Prhl(RL) (BBa_K1529300) in the following experiments and modelings. |
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Fig. 3-2-1-1. 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.(Fig. 3-2-2-1.) 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. 3-2-2-1. The four promoters we tested |
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Fig. 3-2-2-1. shows the fluorescence intensity detected by flow cytometer. Fig. 3-2-2-3. 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- 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. 3-2-2-2. The Fluorescence intensity of the cells (with positive and negative controls) |
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Fig. 3-2-2-3. 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. Replication |
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To confirm the intensity of Prhl(RL) promoter, we did replication study of the reporter assay. |
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We prepared three plasmids shown in below and measured fluorescence intensity by GFP expression when we added signaling molecules. Detail of the protocol of this study is written in the Plux and Prhl promoter assay page. |
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Fig. 3-2-3-1. The fluorescence intensity of the cells with Plux, Prhl and Prhl(RL). |
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Fig. 3-2-3-1. shows Prhl(RL) is still weaker than Plux promoter. Prhl(RL) promoter does not have enough power for our project, so we have to improve Prhl promoter further. |
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4. Materials and methods |
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| 4-1. 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. 3-2-4-1. |
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B. Ptet-RhlR (pSB6A1) Prhl(RR)-GFP (pSB3K3) |
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Fig. 3-2-4-2. |
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C. Ptet-RhlR (pSB6A1) Prhl(LR)GFP (pSB3K3) |
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Fig. 3-2-4-3. |
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D. Ptet-RhlR (pSB6A1) Prhl(RL)-GFP (pSB3K3) |
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Fig. 3-2-4-4. |
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E. Ptet-RhlR (pSB6A1) PlacUV5-GFP (pSB3K3) ...Positive control |
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Fig. 3-2-4-5. |
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F. Ptet-RhlR (pSB6A1) promoter less-GFP(pSB3K3) ...Negative control |
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Fig. 3-2-4-6. |
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| 4-2. Assay Protocol | |
1. Prepare 2 overnight cultures for each sample 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). |
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3. Incubate the fresh cultures in 37°C until the OD590 reaches 0.3. |
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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 | |
| 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 9000x g, 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). | |
5. Reference |
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John S. Chuang et al. (2009) Simpson’s Paradox in a Synthetic Microbial System. SCIENCE 323: 272-275 | |
