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

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Tokyo_Tech

Experiment

C4HSL-dependent 3OC12HSL production

 

Content

1. Introduction

2. Summary of the experiments

3. Results

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

4.2.1. C4HSL-Dependent CmR Expression Assay

4.2.2. C4HSL-Dependent 3OC12HSL Production Assay

5. Reference

 

1. Introduction

 

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. In our bank story, we used signaling molecules C4HSL as money. For construction of the C4HSL-dependent chloramphenicol resistance (CmR) and 3OC12HSL production module, we designed 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 is Company. (Fig. 3-3-1-1.)

 
Fig. 3-3-1-1 Company’s Genetic Circuit
 

In order to confirm Company’s dependency on C4HSL, we measured the growth of Company cell and expression of 3OC12HSL in the presence and absence of C4HSL.

 
 
Fig. 3-3-1-2. C4HSL-depemdent CmR expression Assay Flow Chart
Fig. 3-3-1-3. C4HSL-dependent 3OC12HSL
production Assay Flow Chart
 
 

2. Summary of the experiments

To confirm function of Company cell, we performed two kinds of assays. One is C4HSL-Dependent CmR Expression Assay. In this experiment we prepared four plasmid sets (A, B, C, D) shown in below. (Fig. 3-3-2-1.) Concurrently with C4HSL induction, we added chloramphenicol into the medium containing Company cell and measured optical density for about 8 h to estimate the concentration of the cell. The other is C4HSL-Dependent 3OC12HSL Production Assay. In this experiment we prepared three more plasmid sets (E, F, G) shown in below. (Fig. 3-3-2-1.) First, we added C4HSL to the culture of sender cell, and induced the expression of LasI. Then, the supernatants of the culture were used as the inducer in the reporter assay. We measured the expression of GFP in reporter cells by flow cytometer.

 
Fig.3-3-2-1. Constructed plasmids
 

We prepared four conditions as follow.

 
Sender:
 
A-1) Culture containing Ptet-GFP-Ptet-RhlR(pSB6A1) and Prhl(RL)-CmR-LasI(pSB3K3) cell with C4HSL induction
A-2) Culture containing Ptet-GFP-Ptet-RhlR(pSB6A1) and Prhl(RL)-CmR-LasI(pSB3K3) cell with DMSO (no induction)
 
B-1) Culture containing Ptet-GFP-Ptet-RhlR(pSB6A1) and PlacIq-CmR(pSB3K3) cell with C4HSL induction (C4HSL-Dependent CmR Expression Assay Positive control)
B-2) Culture containing Ptet-GFP-Ptet-RhlR(pSB6A1) and PlacIq-CmR (pSB3K3) cell with DMSO (no induction) (C4HSL-Dependent CmR Expression Assay Positive control)
 
C-1) Culture containing Ptet-GFP-Ptet-RhlR(pSB6A1) and Promoter-less-CmR(pSB3K3) cell with C4HSL induction (C4HSL-Dependent CmR Expression Assay Negative control)
C-2) Culture containing Ptet-GFP-Ptet-RhlR(pSB6A1) and Promoter-less-CmR (pSB3K3) cell with DMSO (no induction) (C4HSL-Dependent CmR Expression Assay Negative control)
 
D-1) Culture containing Ptet-GFP-Ptet-RhlR(pSB6A1) and Plux-CmR(pSB3K3) cell with C4HSL induction (C4HSL-Dependent 3OC12HSL Production Assay Negative control)
D-2) Culture containing Ptet-GFP-Ptet-RhlR(pSB6A1) and Plux-CmR(pSB3K3) cell with DMSO (no induction) (C4HSL-Dependent 3OC12HSL Production Assay Negative control)
 
Reporter:
 
E) Culture containing Ptet-RhlR(pSB6A1) and Prhl(RL)-GFP(pSB3K3) cell
 
F) Culture containing Ptet-RhlR(pSB6A1) and PlacIq-GFP(pSB3K3) cell
 
G) Culture containing Ptet-RhlR(pSB6A1) and Promoter-less-GFP(pSB3K3) cell
 
 
 

3. Result

 

3-1. C4HSL-Dependent CmR Expression Assay

After induction, optical densities were measured to estimate the concentration of the cell. We prepared two types of culture conditions which is different in concentration of chloramphenicol. (Without chloramphenicol and 100 microg/ml)

 
Fig. 3-3-3-1. C4HSL-Dependent Company Growth in no Cm
 
Fig. 3-3-3-2. C4HSL-Dependent Company Growth in 100 microg/mL Cm
 

Fig.3-3-3-1. shows every cell can grow in the absence of chloramphenicol. Conversely, Fig.3-3-3-2. shows some cells cannot grow in presence of chloramphenicol. With induction of C4HSL, the cell containing Prhl(RL)-CmR-LuxI can grow in the presence of chloramphenicol. (The growth curve of the cell is same as the growth curve of positive control.) However, without induction of C4HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol. (The growth curve of the cell is same as the growth curve of negative control.) As a result, only with induction of C4HSL, Prhl(RL)-CmR-LuxI (BBa_K1529302) cell can express CmR and grow well.

 

3-2. C4HSL-Dependent 3OC12HSL Production Assay

Four hours after addition of the supernatants from the culture of sender cells, we measured the expression of GFP in the reporter cells by flow cytometer.

 
Fig. 3-3-3-3. C4HSL-Dependent 3OC12HSL Production Assay result
 

As Fig. 3-3-3-3. shows, when the supernatant of condition ??? was used, the fluorescence intensity of the reporter cell increased. Comparing the results of condition ??? and ???, reporter cell in the supernatant of induced Company cell culture had the ???-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 these experiments, we confirmed that a new part Prhl(RL)-CmR-LasI (BBa_K1529302) synthesized CmR and 3OC12HSL in the presence of C4HSL.

 
 
 

4. Materials and methods

 

4-1 Construction

-Strain

All the samples were JM2.300 strain.

-Plasmids

Sender:
 
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) (C4HSL-Dependent CmR Expression Assay Positive control)
 
Fig. 3-3-4-2.
 
C. Ptet-GFP-Ptet-RhlR (pSB6A1), promoter less CmR (pSB3K3) (C4HSL-Dependent CmR Expression Assay Negative control)
 
Fig. 3-3-4-3.
 
D. Ptet-GFP-Ptet-RhlR (pSB6A1), Plux-CmR (pSB3K3) (C4HSL-Dependent 3OC12HSL Production Assay Negative control)
 
Fig. 3-3-4-4.
 
Reporter:
 
E. Ptet-luxR (pSB6A1), Plux-GFP (pSB3K3)
 
Fig. 3-3-4-5.
 
F. Ptet-LuxR (pSB6A1), PlacIq-GFP (pSB3K3) (Positive control)
 
Fig. 3-3-4-6.
 
G. Ptet-LuxR (pSB6A1), Promoter-less-GFP (pSB3K3) (Negative control)
 
Fig. 3-3-4-7.
 

4-2. Assay Protocol

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4-2-1. C4HSL-Dependent CmR Expression Assay

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. If the OD becomes over 0.5, dilute to 0.5 with LB medium.
3. Add 30 microL of suspension in the following medium.
          1) 3 mL of LB containing Amp and Kan + 3 microL C4HSL (5 microM)
          2) 3 mL of LB containing Amp and Kan + 3 microL DMSO
          3) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100 microg/mL)
                   + 30 microL C4HSL (500 microM)
          4) 3 mL of LB containing Amp, Kan and Cm (final concentration 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 optical density is over 1.0, dilute the cell medium to 1/10.)
 

4-2-2. C4HSL-Dependent 3OC12HSL Production Assay

1. Prepare the supernatant of the sender cell
2. Grow the colony of sender cell in LB containing antibiotic O/N at 37°C.
3. 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.If the OD becomes over 0.5, dilute to o.5 with LB medium.
4. Add 30 microL of suspension in the following medium.
          1) Add 30 microL of 500 microM C4HSL to 3 mL LB containing Amp and Kan
          2) Add 30 microL DMSO to 3 mL of LB containing Amp+Kan
5 .Grow the samples of sender cell at 37°C for 4 hours.
6. Measure optical density every hour. (If optical density is over 1.0, dilute the cell medium to 1/10.)
7. Centrifuge sample at 9000x g, 4°C for 1minute.Filter sterilize supernatant.
8. Use the supernatant in reporter assay.
 
Reporter Assay
1. Grow the colony of Reporter cell (D~F) in LB containing antibiotic(Amp and Kan) over night 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 in OD590 (fresh culture).
3. Add 30 microL of suspension in the following medium.
          1) Filtrate of A①+3mL of LB containing Amp and Kan
          2) Filtrate of A②+3mL of LB containing Amp and Kan
          3) Filtrate of B①+3mL of LB containing Amp and Kan
          4) Filtrate of B②+3mL of LB containing Amp and Kan
          5) Filtrate of C①+3mL of LB containing Amp and Kan
          6) Filtrate of C②+3mL of LB containing Amp and Kan
          7) C4HSL+3mL of LB containing Amp and Kan
          8) DMSO + 3mL of LB containing Amp and Kan
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