Difference between revisions of "Part:BBa K1529321:Design"

(→‎Source)
 
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sequence confirmed
 
sequence confirmed
  
 +
===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 13:28, 23 October 2014

Prhl(RR)_GFP


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 90
    Illegal BamHI site found at 78
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 777


Design Notes

sequence confirmed

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

Composite of BBa_K1529320, BBa_J54103
BBa_K1529320 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