Difference between revisions of "Part:BBa K1529797:Design"
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− | + | ==Design Notes== | |
sequence confirmed | sequence confirmed | ||
+ | ==Materials and Methods== | ||
+ | -Strain<br> | ||
+ | All the samples were JM2.300 strain. | ||
+ | ===3OC12HSL-dependent CmR expression Protocol=== | ||
+ | <b>1.Construction</b><br> | ||
+ | A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)<br> | ||
+ | B. Ptet-LuxR-Plac-RFP(pSB6A1), PlacIq-CmR (pSB3K3) (Positive control)<br> | ||
+ | [[Image:3OC12HSL-dependent_CmR_expression_Construction.png|thumb|center|400px|<b>Fig. 1.</b>Plasmids for the experiment of 3OC12HSL-dependent CmR expression.]] | ||
+ | <b>2.Assay protocol</b><br> | ||
+ | 1.Prepare the overnight culture of cell A and B at 37°C.<br> | ||
+ | 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)<br> | ||
+ | 3. Add 30 microL of suspension in the following medium.<br> | ||
+ | 1) 3 mL of LB containing Amp and Kan + 30 microL C4HSL (final concentration is 500 microM)<br> | ||
+ | 2) 3 mL of LB containing Amp and Kan + 30 microL DMSO<br> | ||
+ | 3) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL C4HSL (final concentration is 500 microM)<br> | ||
+ | 4) 3 mL of LB containing Amp, Kan and Cm (final concentration is 100microg / mL) + 30 microL DMSO<br> | ||
+ | 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.)<br> | ||
− | === | + | |
+ | ===3OC12HSL-dependent C4HSL production Protocol=== | ||
+ | <b>1.Construction</b><br> | ||
+ | <em>Sender</em><br> | ||
+ | A. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR-RhlI(pSB3K3)<br> | ||
+ | B. Ptet-LuxR-Plac-RFP(pSB6A1), Plux-CmR(pSB3K3)...Negative control<br> | ||
+ | <i>Reporter</i><br> | ||
+ | C. Ptet-RhlR(pSB6A1), Plux-GFP(pSB3K3)<br> | ||
+ | D. Ptet-RhlR(pSB6A1), PlacUV5-GFP(pSB3K3)...Positive control<br> | ||
+ | E. Ptet-RhlR(pSB6A1), Promoter-less-GFP(pSB3K3)...Negative control<br> | ||
+ | [[Image:3OC12HSL-dependent_C4HSL_production_Construction.png|thumb|center|600px|<b>Fig. 2.</b>Plasmids for the experiment of 3OC12HSL-dependent C4HSL production.]] | ||
+ | |||
+ | <b>2.Assay protocol</b><br> | ||
+ | Prepare the supernatant of the sender cell<br> | ||
+ | 1. Grow the colony of sender cell in LB containing antibiotic O/N at 37°C.<br> | ||
+ | 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.<br> | ||
+ | 3. Add 30 microL of the culture containing the cells in the following medium.<br> | ||
+ | a) Add 30 microL of 500 microM 3OC12HSL to 3 mL LB containing Amp and Kan<br> | ||
+ | b) Add 30 microL DMSO to 3 mL LB containing Amp and Kan<br> | ||
+ | 4 .Grow the samples of sender cell at 37°C for 8 hours.<br> | ||
+ | 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.<br> | ||
+ | <br> | ||
+ | <em>Reporter Assay</em><br> | ||
+ | 1. Grow the colony of Reporter cell (described upper) in LB containing antibiotic (Amp and Kan) over night at 37°C.<br> | ||
+ | 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).<br> | ||
+ | 3. Add 30 microL of the culture containing reporter cell in the following medium.<br> | ||
+ | 1) 2.7 mL filtrate of Aa +300 microL LB<br> | ||
+ | 2) 2.7 mL filtrate of Ab +300 microL LB<br> | ||
+ | 3) 2.7 mL filtrate of Ba +300 microL LB<br> | ||
+ | 4) 2.7 mL filtrate of Bb +300 microL LB<br> | ||
+ | 5) 3 mL LB + 500 microM C4HSL 30 microM (final concentration is 5 microM)<br> | ||
+ | 6) 3 mL LB + DMSO 30 microL<br> | ||
+ | 4. Grow the samples of Reporter cell in incubator at 37°C for 4 hours.<br> | ||
+ | 5. Start preparing the flow cytometer 1 h before the end of incubation.<br> | ||
+ | 6. After incubation, take the sample, and centrifuge at 9000x g, 1 min, 4°C.<br> | ||
+ | 7. Remove the supernatant by using P1000 pipette.<br> | ||
+ | 8. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3.)<br> | ||
+ | 9. Dispense all of each suspension into a disposable tube through a cell strainer.<br> | ||
+ | 10. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)<br> | ||
+ | |||
+ | |||
+ | ==Source== | ||
Composite of BBa_K395162, BBa_B0034 and BBa_C0070. | Composite of BBa_K395162, BBa_B0034 and BBa_C0070. | ||
Line 21: | Line 79: | ||
[https://parts.igem.org/Part:BBa_C0070 BBa_C0070] : RhlI From kit plate | [https://parts.igem.org/Part:BBa_C0070 BBa_C0070] : RhlI From kit plate | ||
− | + | ==References== | |
1.Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619<br> | 1.Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619<br> | ||
2.Jennifer M. Henke et al. (2004) Bacterial social engagements. TRENDS in Cell Biology 14: 11<br> | 2.Jennifer M. Henke et al. (2004) Bacterial social engagements. TRENDS in Cell Biology 14: 11<br> | ||
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<br> | 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<br> | ||
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<br> | 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<br> |
Latest revision as of 13:42, 23 October 2014
Plux-CmR-RhlI
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1429
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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)
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
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