Difference between revisions of "Part:BBa K1529300:Design"

(→‎Source)
 
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 +
===Materials and Methods===
 +
<b>1. Construction</b><br>
 +
All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.<br>
 +
 +
A. Ptet_RhlR (6A1)　Prhl-GFP (3K3) <br>
 +
B. Ptet_RhlR (6A1)　Prhl(RR)-GFP (3K3) <br>
 +
C. Ptet_RhlR (6A1)　Prhl(LR)-GFP (3K3) <br>
 +
D. Ptet_RhlR (6A1)　Prhl(RL)-GFP (3K3) <br>
 +
E. Ptet_RhlR (6A1)　 Placuv5-GFP (3K3) …positive control<br>
 +
F. Ptet_RhlR (6A1)　promoter less-GFP(3K3) …negative control<br>
 +
 +
[[Image:Improved_Prhl_Promoter_Assay_Construction.png|thumb|center|600px|<b>Fig. 1. </b>Plasmids]]<br>
 +
 +
<b>2. Assay protocol</b><br>
 +
1.Prepare 2 overnight cultures for each sample A~F in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12 h.<br>
 +
2.Dilute the overnight cultures to 1 / 100 in fresh LB medium (3 mL) containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) (→fresh culture).<br>
 +
3.Incubate the fresh cultures in 37°C until the OD590 reaches 0.3.<br>
 +
4.Add 30 microL of 500 microM C4HSL or DMSO as listed below:<br>
 +
　　　A-5 microM: A + C4HSL<br>
 +
　　　A-0 microM: A + DMSO<br>
 +
　　　B-5 microM: B + C4HSL<br>
 +
　　　B-0 microM: B + DMSO<br>
 +
　　　C-5 microM: C + C4HSL<br>
 +
　　　C-0 microM: C + DMSO<br>
 +
　　　D-5 microM: D + C4HSL<br>
 +
　　　D-0 microM: D + DMSO<br>
 +
　　　E-5 microM: E + C4HSL<br>
 +
　　　E-0 microM: E + DMSO<br>
 +
　　　F-5 microM: F + C4HSL<br>
 +
　　　F-0 microM: F + DMSO<br>
 +
5.Incubate the samples at 37°C for 4 h.<br>
 +
6.Start preparing the flow cytometer 1 h before the end of incubation.<br>
 +
7.Take 200 microL of the sample, and centrifuge at 9000x g, 1 min., 4°C.<br>
 +
8.Remove the supernatant by using P1000 pipette.<br>
 +
9.Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.<br>
 +
10.Dispense all of each suspension into a disposable tube through a cell strainer. <br>
 +
11.Measure fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).
  
  

Latest revision as of 13:24, 23 October 2014

Prhl(RL)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Materials and Methods

1. Construction
All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.

A. Ptet_RhlR (6A1)　Prhl-GFP (3K3)
B. Ptet_RhlR (6A1)　Prhl(RR)-GFP (3K3)
C. Ptet_RhlR (6A1)　Prhl(LR)-GFP (3K3)
D. Ptet_RhlR (6A1)　Prhl(RL)-GFP (3K3)
E. Ptet_RhlR (6A1)　 Placuv5-GFP (3K3) …positive control
F. Ptet_RhlR (6A1)　promoter less-GFP(3K3) …negative control

Fig. 1. Plasmids

2. Assay protocol
1.Prepare 2 overnight cultures for each sample A~F in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12 h.
2.Dilute the overnight cultures to 1 / 100 in fresh LB medium (3 mL) containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) (→fresh culture).
3.Incubate the fresh cultures in 37°C until the OD590 reaches 0.3.
4.Add 30 microL of 500 microM C4HSL or DMSO as listed below:
　　　A-5 microM: A + C4HSL
　　　A-0 microM: A + DMSO
　　　B-5 microM: B + C4HSL
　　　B-0 microM: B + DMSO
　　　C-5 microM: C + C4HSL
　　　C-0 microM: C + DMSO
　　　D-5 microM: D + C4HSL
　　　D-0 microM: D + DMSO
　　　E-5 microM: E + C4HSL
　　　E-0 microM: E + DMSO
　　　F-5 microM: F + C4HSL
　　　F-0 microM: F + DMSO
5.Incubate the samples at 37°C for 4 h.
6.Start preparing the flow cytometer 1 h before the end of incubation.
7.Take 200 microL of the sample, and centrifuge at 9000x g, 1 min., 4°C.
8.Remove the supernatant by using P1000 pipette.
9.Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.
10.Dispense all of each suspension into a disposable tube through a cell strainer.
11.Measure fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).


Source

Prhl(RL) promoter was derived from oligo DNA.

References

1.John S. Chuang et al. (2009) Simpson’s Paradox in a Synthetic Microbial System. SCIENCE 323: 272-275
2.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
3.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