Team:Tokyo Tech/Experiment/C4HSL-dependent 3OC12HSL production

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 C4HSL-dependent chloramphenicol resistance (CmR) and 3OC12HSL production module, we constructed a new part Plux-CmR-lasI (BBa_). Plux-CmR-lasI cell is an engineered E.coli that contains a C4HSL-dependent lasI generator and a constitutive rhlR generator. We constructed a new Biobrick part Plux-CmR-lasI by combining Plux-CmR (BBa_K39562) and lasI (BBa_). As a constitutive rhlR generator, we used Pret-rhlR (BBa_S0319). In our bank story, this part is company.

1-1 C4HSL-dependent 3OC12HSL production

First, we performed a reporter assay by using Lux reporter cell to characterize the function of C4HSL-dependent 3OC12HSL production. As the negative control of 3OC12HSL production, we prepared 3OC12HSL non-producer cell. 3OC12HSL non-producer cell contains Plux-CmR instead of Plux-CmR-lasI. The cell of negative control does not produce 3OC12HSL even in the presence of C4HSL.

Sender

Repoter

We prepared four culture conditions as follow.

A) Culture containing Plux-CmR-LasI cell with C4HSL induction

B) Culture containing Plux-CmR-LasI cell without C4HSL induction

C) Culture containing Plux-CmR cell with C4HSL induction

D) Culture containing Plux-CmR cell without C4HSL induction

The supernatants of this four different culture were used as the inducer in the reporter assay.

In the reporter assay, we used a Lux reporter strain that contains Ptet-luxR 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 C4HSL-dependent growth

The cell which contains Plux-CmR-lasI can grow in the medium containing chloramphenicol
(Chloramphenicol is one of the antibiotics. )

After induction, we added chloramphenicol into the medium and measured optical density
after induction.

2. Results

2-1 C4HSL-dependent 3OC12HSL production

We measured the expression of GFP in the reporter cell by flow cytometer.

2-2 C4HSL-dependent growth

After induction, optical density were measured to estimate the concentration of the cell.

Fig. 3-1-1.

Fig. 3-1-2.

explanation?

3. Materials and methods

3-1 Construction

-Strain

All the samples were JM2.300 strain

-Plasmids

3-2

3-2-1. C4HSL-dependent 3OC12HSL 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 37C 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 30µL of 500µM C12HSL to 3mL LB containing Amp and Kan

Add 30µL 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.)

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 37°C 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

3OC12HSL＋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. C4HSL-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 3microL 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.)

Team:Tokyo Tech/Experiment/C4HSL-dependent 3OC12HSL production

From 2014.igem.org

Experiment

1. Summary of the experiment

2. Results

3. Materials and methods

4. Reference