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Experiment
3OC12HSL-dependent C4HSL production module |
1. Summary of the experiment |
Construction of the 3OC12HSL-dependent C4HSL production and chloramphenicol resistance expression module |
We created a symbiosis of Company E.coli and Customer E.coli for reproducing the situation in real economy. We used signaling molecules and antibiotics resistance gene ,and constructed signal-dependent signal production in our system. |
For construction of the 3OC12HSL-dependent chloramphenicol resistance (CmR) and C4HSL production module, we constructed a new part Plux-CmR-rhlI (BBa_K1529265). Plux-CmR-RhlI cell is an engineered E.coli that contains a 3OC12HSL-dependent rhlI generator and a constitutive luxR generator. We constructed a new Biobrick part Plux-CmR-rhlI by combining Plux-CmR (BBa_K39562) and rhlI (BBa_). As a constitutive luxR generator, we used Pret-luxR (BBa_S0319). In our bank story, this part is customer. |
1-1 3OC12HSL-dependent C4HSL production |
First, we performed a reporter assay by using rhl reporter cell to characterize the function of 3OC12HSL-dependent C4HSL production. As the negative control of C4HSL production, we prepared C4HSL non-producer cell. C4HSL non-producer cell contains PlacIq-CmR instead of Plux-CmR-rhlI. The cell of negative control does not produce C4HSL even in the presence of 3C12HSL. |
Sender |
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Repoter |
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We prepared four culture conditions as follow. |
| A) Culture containing Plux-CmR-RhlI cell with 3OC12HSL induction |
| B) Culture containing Plux-CmR-RhlI cell without 3OC12HSL induction |
| C) Culture containing Plux-CmR cell with 3OC12HSL induction |
| D) Culture containing Plux-CmR cell without 3OC12HSL induction |
The supernatants of this four different culture were used as the inducer in the reporter assay. |
In the reporter assay, we used a Rhl reporter strain that contains Ptet-rhlR and Plux-GFP. Also, a reporter cell that expresses GFP constitutively and a reporter cell that does not express GFP were used as the positive control and the negative control, respectively. |
1-2 3OC12HSL-dependent growth |
The cell which contains Plux-CmR-rhlI can grow in the medium even containing chloramphenicol .( Chloramphenicol is one of the antibiotics. ) |
After induction, we added chloramphenicol into the medium and measured optical density hourly after induction. |
2. Results |
2-1 3OC12HSL-dependent C4HSL production |
We measured GFP expression in the reporter cell by flow cytometer |
2-2 3OC12HSL-dependent growt |
After induction, optical density were measured to estimate the concentration of the cell. |
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Fig. 3-2-1 |
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Fig. 3-2-2 |
As Fig 3-2-1 shows, the cell containing Plux-CmR-RhlI can grow with induction of 3OC12SL, but can’t without induction. Plux-CmR-RhlI cell grows more slowly than the positive control. The amount of CmR is lower than the positive control, Compared Fig 3-2-1 to Fig 3-2-2, With Cm, the cell grow more slowly than without Cm. geExpression of CmR in Plux-CmR-RhlI depende on induction by adding 2OC12HSL. |
3. Materials and methods |
3-1 Construction |
-Strain |
All the samples were JM2.300 strain |
-Plasmids |
3-2 Protocol |
3-2-1. 3OC12HSL-dependent C4HSL production assay by using reporter assay |
Prepare the supernatant of the sender cell |
| 1. Grow the colony of sender cell in LB containing antibiotic O/N at 37°C. |
| 2. Make a 1:100 dilution in 3 mL of fresh LB containing antibiotic and grow the cells at 37°C until the observed OD590 reaches 0.5. |
| 3. Centrifuge 1mL of the sample at 5000g, RT for 1 minute. |
| 4. Suspend the pellet in 1 mL of LB containing Ampicillin（50μg/mL）and Kanamycin (30μg/mL) |
| 5. Add 30µL of suspension in the following medium. |
| Add 3µL of 5µM C12HSL to 3mL LB containing Amp and Kan |
| Add 3µL DMSO to 3 µM of LB containing Amp and Kan |
| 6. Grow the samples of sender cell at 37°C for 4 hours. |
| 7. Measure optical density every hour (If optical density is over 1.0, dilute the cell medium.) |
| 8. Centrifuge sample at 9000g, 4°C for 1minute.Filter sterilize supernatant. |
| 9. Use the supernatant in reporter assay |
Reporter Assay |
| 1. Grow the colony of Reporter cell(D～F) in LB containing antibiotic O/N at 37°C. |
| 2. Make a 1:100 dilution in 3 mL of fresh LB+ antibiotic and grow the cells at 37C until you reach an 0.5 OD590.(fresh culture) |
| 3. Centrifuge sample at 5000g, 25°C RT for 1 minute. Discard the supernatant. |
| 4. Suspended the sample in 3 mL of LB containing Ampicillin(50μg/mL) and Kanamycin(30μg/mL) |
| 5. Add 30µL of suspension in the following medium. |
| Filtrate of A①＋3mL of LB containing Amp and Kan |
| Filtrate of A②＋3mL of LB containing Amp and Kan |
| Filtrate of B①＋3mL of LB containing Amp and Kan |
| Filtrate of B②＋3mL of LB containing Amp and Kan |
| Filtrate of C①＋3mL of LB containing Amp and Kan |
| Filtrate of C②＋3mL of LB containing Amp and Kan |
| C4HSL＋3mL of LB containing Amp and Kan |
| DMSO + 3mL of LB containing Amp and Kan |
| 6. Grow the samples of Reporter cell in incubator at 37°C for 4 hours. |
| 7. Start preparing the flow cytometer 1 h before the end of incubation. |
| 8. Take 200 microL of the sample, and centrifuge at 9000 Xg, 1 min, 4°C. |
| 9. Remove the supernatant by using P1000 pipette. |
| 10. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. |
| 11. Dispense all of each suspension into a disposable tube through a cell strainer. |
| 12. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company). |
3-2-2. 3OC12HSL-depemdent CmR expression |
| 1. Grow the colony of sender cell in LB containing antibiotic O/N at 37°C. |
| 2. Make a 1:100 dilution in 3 mL of fresh LB containig antibiotic and grow the cells at 37°C until the observed OD590 reaches 0.5.(fresh culture) |
| 3. Centrifuge 1mL of the sample at 5000g, RT for 1 minute. |
| 4. Suspend the pellet in 1 mL of LB containing Ampicillin（50 microg/mL）and Kanamycin(30 microg/mL) |
| 5. Add 30µL of suspension in the following medium. |
| Add 3µL of 5µM C12HSL to 3mL LB containing Amp,Kan(concentration is described upper) and Cm(100 microg /mL) |
| Add 3µ DMSO to 3mL of LB containing Amp and Kan |
| 6. Grow the samples of sender cell at 37°C for 4 hours. |
| 7. Measure optical density every hour. (If optical density is over 1.0, dilute the cell medium.) |
4. Reference |
