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

 
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<partinfo>BBa_K1529301 short</partinfo>
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__TOC__
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===Materials and Methods===
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<b>1. Construction</b><br>
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All the samples were JM2.300 strain with antibiotic resistance to ampicillin and kanamycin.<br>
  
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__NOTOC__
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A. Ptet_RhlR (6A1)　Prhl_GFP (3K3) #1<br>
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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B. Ptet_RhlR (6A1)　Prhl_GFP (3K3) #2<br>
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how you used this part and how it worked out.
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C. Ptet_RhlR (6A1)　Prhl(LR)_GFP (3K3) #1<br>
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D. Ptet_RhlR (6A1)　Prhl(LR)_GFP (3K3) #2<br>
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E. Ptet_RhlR (6A1)　Prhl(RL)_GFP (3K3) #1<br>
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F. Ptet_RhlR (6A1)　Prhl(RL)_GFP (3K3) #2<br>
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G. Ptet_RhlR (6A1)　Prhl_a_GFP (3K3) #1<br>
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H. Ptet_RhlR (6A1)　Prhl_a_GFP (3K3) #2<br>
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I. Ptet_RhlR (6A1)　 Placuv5_GFP (3K3) #1…positive control<br>
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J. Ptet_RhlR (6A1)　ΔP_GFP(3K3) #1…negative control<br>
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[[Image:titech2013_parts_K1139150_exp_Fig1.jpg|frameless|none|250px||]]
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<b>2. Assay protocol</b><br>
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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.<br>
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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.<br>
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3.Incubate the fresh cultures in 37°C until the observed OD590 reaches 0.3 (Actual value 0.42).<br>
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4.Add 30 microL of 500 microM C4HSL or DMSO as listed below:<br>
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A-5 μM: A + C4HSL<br>
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A-0 μM: A + DMSO<br>
 +
B-5 μM: B + C4HSL<br>
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B-0 μM: B + DMSO<br>
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C-5 μM: C + C4HSL<br>
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C-0 μM: C + DMSO<br>
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D-5 μM: D + C4HSL<br>
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D-0 μM: D + DMSO<br>
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E-5 μM: E + C4HSL<br>
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E-0 μM: E + DMSO<br>
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F-5 μM: F + C4HSL<br>
 +
F-0 μM: F + DMSO<br>
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G-5 μM: G + C4HSL<br>
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G-0 μM: G + DMSO<br>
 +
H-5 μM: H + C4HSL<br>
 +
H-0 μM: H + DMSO<br>
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I-5 μM: H + C4HSL <br>
 +
I-0 μM: I + DMSO<br>
 +
J-5 μM: H + C4HSL <br>
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J-0 μM: J + DMSO<br>
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5.Incubate the samples at 37°C for 4 h.<br>
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6.Start preparing the flow cytometer 1 h before the end of incubation.<br>
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7.Take 200 microL of the sample, and centrifuge at 9000 Xg, 1 min, 4°C.<br>
 +
8.Remove the supernatant by using P1000 pipette.<br>
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9.Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.<br>
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10.Dispense all of each suspension into a disposable tube through a cell strainer.<br>
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11.Measure fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).<br>
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===Results===
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Fig. 1 shows the fluorescence intensity detected by flow cytometer.<br>
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[[Image:titech2014_parts_K1529301_Fig2.jpg|thumb|center|310px|<b>Fig. 1.</b> Fluorescence intensity detected by flow cytometer]]<br>
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<br>
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For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/RM-lac_Hybrid_Promoter_Assay our work in Tokyo_Tech 2013 wiki].
  
 
===Applications of BBa_K1529301===
 
===Applications of BBa_K1529301===
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Revision as of 11:06, 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

Titech2013 parts K1139150 exp Fig1.jpg

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 shows the fluorescence intensity detected by flow cytometer.


File:Titech2014 parts K1529301 Fig2.jpg
Fig. 1. Fluorescence intensity detected by flow cytometer


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

Applications of BBa_K1529301

User Reviews

UNIQ60e58b995ada5633-partinfo-00000001-QINU UNIQ60e58b995ada5633-partinfo-00000002-QINU