Difference between revisions of "Part:BBa K1529301:Experience"

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===Results===
 
===Results===
[[Image:Improved_Prhl_Promoter_Assay_Result|thumb|center|250px|<b>Fig. 1.</b> The fluorescence intensity of the cells (with positive and negative controls)]]
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[[Image:Improved_Prhl_Promoter_Assay_Result.png|thumb|center|600px|<b>Fig. 1.</b> The fluorescence intensity of the cells (with positive and negative controls)]]
  
  
[[Image:Improved_Prhl_Promoter_Assay_Extracted_Result|thumb|center|250px|<b>Fig. 2.</b> The fluorescence intensity of the cells with the original Prhl (BBa_R0071), Prhl(RR) (BBa_K1529320), and Prhl(RL) (BBa_K1529300)]]
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[[Image:Improved_Prhl_Promoter_Assay_Extracted_Result.png|thumb|center|600px|<b>Fig. 2.</b> The fluorescence intensity of the cells with the original Prhl (BBa_R0071), Prhl(RR) (BBa_K1529320), and Prhl(RL) (BBa_K1529300)]]
  
 
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===Discussion===
 
===Discussion===
 
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Revision as of 14:39, 13 October 2014

Prhl(RL)-GFP

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(LR)_GFP (3K3)
C. Ptet_RhlR (6A1) Prhl(RL)_GFP (3K3)
D. Ptet_RhlR (6A1) Prhl_a_GFP (3K3)
E. Ptet_RhlR (6A1)  Placuv5_GFP (3K3) …positive control
F. Ptet_RhlR (6A1) ΔP_GFP(3K3) …negative control


2. Assay protocol
1.Prepare overnight cultures of every samples A~J in LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12 h.
2.Dilute the overnight culture to 1 / 100 in fresh LB medium (3 mL) containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL). (→fresh culture) Make glycerol stocks from the remainders.
3.Incubate the fresh cultures in 37°C until the observed OD590 reaches 0.3 (Actual value 0.42).
4.Add 30 microL of 500 microM C4HSL or DMSO as listed below:
A-5 μM: A + C4HSL
A-0 μM: A + DMSO
B-5 μM: B + C4HSL
B-0 μM: B + DMSO
C-5 μM: C + C4HSL
C-0 μM: C + DMSO
D-5 μM: D + C4HSL
D-0 μM: D + DMSO
E-5 μM: E + C4HSL
E-0 μM: E + DMSO
F-5 μM: F + C4HSL
F-0 μM: F + DMSO
G-5 μM: G + C4HSL
G-0 μM: G + DMSO
H-5 μM: H + C4HSL
H-0 μM: H + DMSO
I-5 μM: H + C4HSL
I-0 μM: I + DMSO
J-5 μM: H + C4HSL
J-0 μM: J + 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 9000 Xg, 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).


Results

Fig. 1. The fluorescence intensity of the cells (with positive and negative controls)


Fig. 2. The fluorescence intensity of the cells with the original Prhl (BBa_R0071), Prhl(RR) (BBa_K1529320), and Prhl(RL) (BBa_K1529300)


Discussion





For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/RM-lac_Hybrid_Promoter_Assay our work in Tokyo_Tech 2013 wiki].

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