From 2014.igem.org

We designed signal-dependent signal production in our system by using signaling molecules and antibiotics resistance gene. In our bank story, we used signaling molecules 3OC12HSL as products.

For construction of the 3OC12HSL-dependent chloramphenicol resistance (CmR) and C4HSL production module, we constructed a new part Plux-CmR-RhlI (BBa_K1529797). Plux-CmR-RhlI cell is an engineered E. coli that contains a 3OC12HSL-dependent RhlI generator and a constitutive LuxR generator. As a constitutive LuxR generator, we used Ptet-LuxR. In our bank story, this part imitates the function of Customer. (Fig. 3-4-1-1.)　We confirmed that 3OC12HSL-dependent growth by measuring the optical density, and 3OC12HSL-dependent C4HSL production by using reporter cell.

Fig. 3-4-1-1. Genetic Circuit of Customer E. coli

2. Summary of the experiments

2-1. 3OC12HSL-dependent CmR expression

Fig. 3-4-2-1. 3OC12HSL-dependent CmR expression assay Flow Chart

We confirmed the function of 3OC12HSL-dependent CmR expression by measuring optical density of the cell cultures containing chloramphenicol.

In this experiment we prepared two plasmids, A and B. (See Fig. 3-4-2-2.) Right after the 3OC12HSL induction, we added chloramphenicol into the medium containing Customer cell. We measured optical density for about 8 h to estimate the concentration of the cell.

Fig. 3-4-2-2. Plasmids for the experiment of 3OC12HSL-dependent CmR expression

2-2. 3OC12HSL-dependent C4HSL production

Fig. 3-4-2-3. 3OC12HSL-dependent C4HSL production assay Flow Chart

We performed a reporter assay by using reporter cell C, D and E to characterize the function of 3OC12HSL-dependent C4HSL production. Plux-CmR-RhlI cell containing constitutive LuxR generator express RhlI and produce C4HSL (RhlI) in the presence of 3OC12HSL. Since C4HSL is excreted to the culture, the supernatant of the sender cell contains C4HSL when the part works as expected.

Reporter cells was incubated in the supernatant of the culture of sender cell. When there are C4HSL in the supernatant, reporter cell expresses GFP. We checked the fluorescence intensity of reporter cell to confirm of the production of C4HSL.The fluorescence intensity of the reporter cell was measured by Flow Cytometer.

Fig. 3-4-2-4. Plasmids for the experiment of 3OC12HSL-dependent C4HSL production

We prepared the following conditions for the induction of reporter cells. (PlacIq-CmR cell was used as the negative control of RhlI.)

(1)Culture containing sender A (Plux-CmR-RhlI) with 3OC12HSL induction

(2) Culture containing sender A (Plux-CmR-RhlI) without induction

(3) Culture containing sender B (PlacIq-CmR) with 3OC12HSL induction

(4)Culture containing sender B (PlacIq-CmR) without induction

(5)5 microM of artificial C4HSL in LB medium

(6)DMSO in LB medium

Reporter cells

C. The cell containing constitutive RhlR generator and Prhl(RL)-GFP

D. The cell containing constitutive RhlR generator and PlacUV5-GFP…Positive control

E. The cell containing constitutive RhlR generator and Promoter-less-GFP…Negative control

3. Results

3-1. 3OC12HSL-depemdent CmR expression assay

We tested two types of culture condition which contains different concentration of chloramphenicol(Cm). (0 and 100 microg / mL)

Fig.3-4-3-1. and Fig.3-4-3-2.show the condition in the absence and the presence of chloramphenicol, respectively.

Fig.3-4-3-1. shows that every cell can grow in the absence of chloramphenicol.

Fig. 3-4-3-1. 3OC12HSL-dependent customer growth in no chloramphenicol

Fig.3-4-3-2. 3OC12HSL-dependent Customer growth in 100 microg / mL chloramphenicol

On the other hand, in the presence of chloramphenicol, the cell containing Plux-CmR-RhlI can grow only when induced by 3OC12HSL. Without the induction of 3OC12HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol. As a result, we confirmed that Plux-CmR-RhlI expressed CmR when induced by 3OC12HSL as expected.

3-2. 3OC12HSL-dependent C4HSL production assay

Fig. 3-4-3-3.shows the fluorescence intensities generated by reporter cells. When the reporter cell C (Plux-CmR-RhlI) was incubated in the condition (1) (the culture of the induced Customer cell), the fluorescence intensity of the reporter cell increased. Comparing the results of condition (1) and (2), reporter cell in the supernatant of (1) had 95-fold higher fluorescence intensity.

This result indicates that Customer cell produced C4HSL in response to 3OC12HSL induction by the function of Plux-CmR-RhlI.

From this experiment, we confirmed that a new part Plux-CmR-RhlI synthesized C4HSL (RhlI) as expected.

Fig.3-4-3-3. Customer excretes C4HSL when C12HSL exists

4. Materials and methods

4-1 Construction

-Strain

All the samples were JM2.300 strain.

-Plasmids

3OC12HSL-dependent CmR expression

A.Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)

Fig. 3-4-4-1.

B. Ptet-GFP-Ptet-RhlR (pSB6A1), PlacIq-CmR (pSB3K3)...Positive control

Fig. 3-4-4-2.

3OC12HSL-dependent C4HSL production

Sender:

A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)

Fig. 3-4-4-3.

B. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR(pSB3K3)...Negative control

Fig. 3-4-4-4.

Reporter:

C. Ptet-RhlR(pSB6A1), Plux-GFP(pSB3K3)

Fig. 3-3-4-5.

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

Fig. 3-4-4-6.

E. Ptet-RhlR(pSB6A1),　Promoter-less-GFP(pSB3K3)...Negative control

Fig. 3-4-4-7.

4-2. Assay Protocol

4-2-1. 3OC12HSL-depemdent CmR expression

1.Prepare the overnight culture of cell A and B at 37°C.

2.Make a 1:100 dilution in 3 mL of fresh LB containing antibiotics and grow the cell at 37°C until the observed OD590 reaches 0.5 (→fresh culture)

3. Add 30 microL of suspension in the following medium.

1) 3 mL of LB containing Amp and Kan + 30 microL C4HSL (final concentration is 500 microM)

2) 3 mL of LB containing Amp and Kan + 30 microL DMSO

3) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL C4HSL (final concentration is 500 microM)

4) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL DMSO

4. Grow the samples of sender cells at 37°C for more than 10 hours. Measure optical density every hour. (If optical density is over 1.0, dilute the cell medium.)

4-2-2. 3OC12HSL-dependent C4HSL production

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. Add 30 microL of the culture containing the cells in the following medium.

a) Add 30 microL of 500 microM 3OC12HSL to 3 mL LB containing Amp and Kan

b) Add 30 microL DMSO to 3 mL LB containing Amp and Kan

4 .Grow the samples of sender cell at 37°C for 8 hours.

5. Centrifuge sample at 9000x g, 4°C for 1minute. Filter sterilize supernatant. (Pore size is 0.22 microm. ) Use this supernatant in reporter assay.

Reporter Assay

1. Grow the colony of Reporter cell (described upper) in LB containing antibiotic (Amp and Kan) over night at 37°C.

2. Make a 1:100 dilution in 3 mL of fresh LB+ antibiotics and grow the cells at 37°C until you reach an 0.5 in OD590 (fresh culture).

3. Add 30 microL of the culture containing reporter cell in the following medium.

1) 2.7 mL filtrate of Aa +300 microL LB

2) 2.7 mL filtrate of Ab +300 microL LB

3) 2.7 mL filtrate of Ba +300 microL LB

4) 2.7 mL filtrate of Bb +300 microL LB

5) 3 mL LB + 500 microM C4HSL 30 microM (final concentration is 5 microM)

6) 3 mL LB + DMSO 30 microL

4. Grow the samples of Reporter cell in incubator at 37°C for 4 hours.

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

6. After incubation, take the sample, and centrifuge at 9000x g, 1 min, 4°C.

7. Remove the supernatant by using P1000 pipette.

8. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3.)

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

10. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)

5. Reference

1. Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619

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