Part:BBa_K1529302:Experience

Prhl(RL)-CmR-LasI

Materials and Methods

-Strain
All the samples were JM2.300 strain.

C4HSL-dependent CmR expression Protocol

1.Construction

A. Ptet-GFP-Ptet-RhlR (pSB6A1), Prhl(RL)-CmR-LasI(pSB3K3)
B. Ptet-GFP-Ptet-RhlR (pSB6A1), PlacIq-CmR (pSB3K3)…Positive control
C. Ptet-GFP-Ptet-RhlR (pSB6A1), promoter less CmR (pSB3K3)… Negative control

2.Assay protocol

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.)

C4HSL-dependent 3OC12HSL production Protocol

1.Construction

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

Reporter
E. Ptrc-LasR (pSB6A1), Plas-GFP (pSB3K3)
F. Ptet-LuxR (pSB6A1), PlacIq-GFP (pSB3K3)...Positive control
G. Ptet-LuxR (pSB6A1), Promoter-less-GFP (pSB3K3)...Negative control

2.Assay protocol
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.)

Results

C4HSL-dependent CmR expression Result

We tested two types of culture condition which contains different concentration of chloramphenicol(Cm). (0 and 100 microg / mL)
Fig. 1, Fig. 2 shows the condition in the absence and presence of chloramphenicol, respectively.

• 

  Fig. 1. C4HSL-dependent CmR expressionh in no chloramphenicol

• 

  Fig. 2. C4HSL-dependent CmR expressionh in 100 microg / mL chloramphenicol

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

C4HSL-dependent 3OC12HSL production Result

Fig. 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. 3OC12HSL production in the presence of C4HSL

User Reviews

UNIQ6631dca3c51c8b44-partinfo-00000002-QINU UNIQ6631dca3c51c8b44-partinfo-00000003-QINU