Difference between revisions of "Part:BBa K1529301:Experience"
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[[Image:titech2014_parts_K1529301_Fig2.jpg|thumb|center|310px|<b>Fig. 2.</b> Fluorescence intensity detected by flow cytometer]]<br>
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[[Image:titech2014_parts_GFPassay_main_Fig2.jpg|thumb|center|310px|<b>Fig. 2.</b> Fluorescence intensity detected by flow cytometer]]
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===Discussion===
 
===Discussion===

Revision as of 12:02, 3 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) #1
B. Ptet_RhlR (6A1)　Prhl_GFP (3K3) #2
C. Ptet_RhlR (6A1)　Prhl(LR)_GFP (3K3) #1
D. Ptet_RhlR (6A1)　Prhl(LR)_GFP (3K3) #2
E. Ptet_RhlR (6A1)　Prhl(RL)_GFP (3K3) #1
F. Ptet_RhlR (6A1)　Prhl(RL)_GFP (3K3) #2
G. Ptet_RhlR (6A1)　Prhl_a_GFP (3K3) #1
H. Ptet_RhlR (6A1)　Prhl_a_GFP (3K3) #2
I. Ptet_RhlR (6A1)　 Placuv5_GFP (3K3) #1…positive control
J. Ptet_RhlR (6A1)　ΔP_GFP(3K3) #1…negative control

File:Titech2014 parts K1529301 exp Fig1.jpg
Fig. 2. タイトル名

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. 2 shows the fluorescence intensity detected by flow cytometer.


File:Titech2014 parts GFPassay main Fig2.jpg
Fig. 2. Fluorescence intensity detected by flow cytometer


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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