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



1. Introduction
We designed signal-dependent signal production in our system by using signaling molecules and antibiotics resistance gene. In our bank story, we used signaling molecule C4HSL as money.
For construction of the C4HSL-dependent chloramphenicol resistance gene product(CmR) and 3OC12HSL production module, we constructed a new part Prhl(RL)-CmR-LasI(BBa_K1529302). Prhl(RL)-CmR-LasI cell is an engineered E. coli that contains a C4HSL-dependent LasI generator and a constitutive RhlR generator. As a constitutive RhlR generator, we used Ptet-RhlR. In our bank story, this part imitates the functions of Company. (Fig. 3-3-1-1) We confirmed the C4HSL-dependent growth by measuring optical density, and C4HSL-dependent 3OC12HSL production by using reporter cell.


Fig. 3-3-1-1. Genetic Circuit of Company E. coli




2. Summary of the experiments
2-1. C4HSL-dependent CmR expression


Fig. 3-3-2-1. Flow chart of C4HSL-dependent CmR expression assay

We confirmed the function of C4HSL-dependent CmR expression by measuring optical density of the cultures containing chloramphenicol.
In this experiment we prepared three plasmids, A, B and C. (See Fig. 3-3-2-2) Right after the C4HSL induction, we added chloramphenicol into the medium containing Company cell. We measured the optical density for about eight hours to estimate the concentration of the cell.


Fig. 3-3-2-2. Plasmids for the experiment of C4HSL-dependent CmR expression

2-2. C4HSL-dependent 3OC12HSL production

We performed a reporter assay by using reporter cells to characterize the function of C4HSL-dependent C4HSL production. Prhl(RL)-CmR-LasI cell containing constitutive RhlR generator expresses LasI and produces 3OC12HSL in the presence of C4HSL. Since 3OC12HSL is excreted to the culture, the supernatant of the sender cell contains 3OC12HSL when the part works as expected.
Reporter cells are incubated in the supernatant of the culture of sender cells. When there are 3OC12HSL in the supernatant, reporter cell expresses GFP. We checked the fluorescence of reporter cells to confirm the expression of 3OC12HSL. The expression of the reporter cells were measured by flow cytometer.(See Fig.3-3-2-3)


Fig. 3-3-2-3. Flow chart of C4HSL-dependent 3OC12HSL production Assay

Fig. 3-3-2-4.Plasmids for the experiment of C4HSL-dependent 3OC12HSL production
We prepared the following conditions for the induction of reporter cells. (Plux-CmR cell was used as the negative control. See Fig. 3-3-2-4)
(1) Culture containing Prhl(RL)-CmR-lasI cell with C4HSL induction
(2) Culture containing Prhl(RL)-CmR-lasI cell without induction
(3) Culture containing Plux-CmR cell with C4HSL induction
(4) Culture containing Plux-CmR cell without induction
(5) 5 microM of synthetic C4HSL in LB medium
(6) DMSO in LB medium

Reporter
E) The cell containing constitutive LasR generator and Plas-GFP cell
F) The cell containing constitutive LuxR generator and PlacIq-GFP cell…Positive control
G) The cell containing constitutive LuxR generator and Promoter-less-GFP cell…Negative control



3. Result

3-1. C4HSL-dependent CmR expression assay
We tested two types of culture condition which contains different concentration of chloramphenicol(Cm). (0 and 100 microg / mL)
Fig.3-3-3-1, Fig.3-3-3-2 shows the condition in the absence and presence of chloramphenicol, respectively.
Fig 3-3-3-1. shows that every cell can grow in the absence of chloramphenicol.


Fig. 3-3-3-1. C4HSL-Dependent Company growth with no Cm addition


Fig. 3-3-3-2. C4HSL-Dependent Company Growth in 100 microg/mL Cm

On the other hand, in the presence of chloramphenicol, the cell containing Prhl(RL)-CmR-LasI can grow only when induced by C4HSL. Without the induction of C4HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol. As a result, we confirmed that Prhl(RL)-CmR-LasI expressed CmR when induced by C4HSL as expected.

3-2. C4HSL-dependent 3OC12HSL Production Assay
Fig. 3-3-3-3 shows the fluorescence intensities generated by the reporter cells. When the reporter cell E was incubated in the condition (1) (the culture of the induced Company 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 29-fold higher fluorescence intensity.

This result indicates that Company cell produced 3OC12HSL in response to C4HSL induction by the function of Prhl(RL)-CmR-LasI.

From this experiment, we confirmed that a new part Prhl(RL)-CmR-LasI synthesized 3OC12HSL (LasI) as expected.

Fig. 3-3-3-3. Company excretes 3OC12HSL when C4HSL exists




4. Materials and methods

4-1 Construction
-Strain
All the samples were JM2.300 strain.
-Plasmids
--C4HSL-dependent CmR expression

A. Ptet-GFP-Ptet-RhlR (pSB6A1), Prhl(RL)-CmR-LasI(pSB3K3)


Fig. 3-3-4-1.

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


Fig. 3-3-4-2.

C. Ptet-GFP-Ptet-RhlR (pSB6A1), promoter less CmR (pSB3K3)… Negative control


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


Sender
A. Ptet-GFP-Ptet-RhlR (pSB6A1), Prhl(RL)-CmR-LasI (pSB3K3)


Fig. 3-3-4-4.

D. Ptet-GFP-Ptet-RhlR (pSB6A1), Plux-CmR (pSB3K3)


Fig. 3-3-4-5.

Reporter

E. Ptrc-LasR (pSB6A1), Plas-GFP (pSB3K3)


Fig. 3-3-4-6.

F. Ptet-LuxR (pSB6A1), PlacIq-GFP (pSB3K3)...Positive control


Fig. 3-3-4-7.

G. Ptet-LuxR (pSB6A1), Promoter-less-GFP (pSB3K3)...Negative control


Fig. 3-3-4-8.

4-2. Assay Protocol
4-2-1. C4HSL-Dependent CmR Expression Assay
1. Prepare overnight cultures for the sender cells in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12h.
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.(→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 5 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 100 microg/mL) + 30 microL C4HSL (final concentration is 500 microM)
4) 3 mL of LB containing Amp, Kan and Cm (final concentration of Cm is 100 microg/mL) + 30 microL DMSO
4. Grow the samples of sender cells at 37°C for more than 8 hours.
5. Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/10.)

4-2-2. C4HSL-Dependent 3OC12HSL Production Assay
Prepare the supernatant of the sender cell
1. Prepare overnight cultures for the sender cells in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12h.
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 15 microL of 10 mM C4HSL to 3 mL LB containing Amp and Kan (final concentration is 50 microM)
b) Add 15 microL DMSO to 3 mL of LB containing Amp＋Kan
4. Grow the samples of sender cell at 37°C for 8 hours.
5. Measure the optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/10.)
6. Centrifuge the sample at 9000x g, 4°C for 1 min. Filter sterilize the supernatant. (Pore size is 0.22 microm.)
7. Use the supernatant in reporter assay.

Reporter Assay
1. Prepare overnight cultures for the Reporter cell (E～G) in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12h.
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 in OD590 (fresh culture).
3. Add 30 microL of suspension 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 Da +300 microL LB
4) 2.7 mL filtrate of Db +300 microL LB
5) 3 mL LB + 5 microM C12HSL 3 microL (Final concentration is 5 nM)
6) 3 mL LB + DMSO 3 microL
4. Grow the samples of Reporter cell in incubator at 37°C for 4 h.
5. Start preparing the flow cytometer 1 h before the end of incubation.
6. After the 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

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

From 2014.igem.org

Experiment

1. Introduction

2. Summary of the experiments

2-1. C4HSL-dependent CmR expression

2-2. C4HSL-dependent 3OC12HSL production

3. Result

3-1. C4HSL-dependent CmR expression assay

3-2. C4HSL-dependent 3OC12HSL Production Assay

4. Materials and methods

4-1 Construction

4-2. Assay Protocol

5. Reference