Difference between revisions of "Part:BBa K1529797:Design"

Latest revision as of 13:42, 23 October 2014

Plux-CmR-RhlI

Design Notes

sequence confirmed

Materials and Methods

-Strain
All the samples were JM2.300 strain.

3OC12HSL-dependent CmR expression Protocol

1.Construction
A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)
B. Ptet-LuxR-Plac-RFP(pSB6A1), PlacIq-CmR (pSB3K3) (Positive control)

Fig. 1.Plasmids for the experiment of 3OC12HSL-dependent CmR expression.

2.Assay protocol
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.)

3OC12HSL-dependent C4HSL production Protocol

1.Construction
Sender
A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)
B. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR(pSB3K3)...Negative control
Reporter
C. Ptet-RhlR(pSB6A1), Plux-GFP(pSB3K3)
D. Ptet-RhlR(pSB6A1), PlacUV5-GFP(pSB3K3)...Positive control
E. Ptet-RhlR(pSB6A1), Promoter-less-GFP(pSB3K3)...Negative control

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

2.Assay protocol
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.)

Source

Composite of BBa_K395162, BBa_B0034 and BBa_C0070.

BBa_K395162 : Lux-promoter_CmR From Tokyo_Tech 2010

BBa_B0034 : RBS From kit plate

BBa_C0070 : RhlI From kit plate

References

1.Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619
2.Jennifer M. Henke et al. (2004) Bacterial social engagements. TRENDS in Cell Biology 14: 11
3.Gabriella Pessi et al. (2000) Transcriptional Control of the Hydrogen Cyanide Biosynthetic Genes hcnABC by the Anaerobic Regulator ANR and the Quorum-Sensing Regulators LasR and RhlR in Pseudomonas aeruginosa Journal of Bacteriology 182(24): 6940–6949
4.Kendall M. Gray et al. (1994) Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. Journal of Bacteriology 176(10): 3076–3080