Difference between revisions of "Part:BBa K1529301:Experience"
Line 1: | Line 1: | ||
+ | <partinfo>BBa_K1529301 short</partinfo> | ||
+ | __TOC__ | ||
+ | ===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) #1<br> | |
− | + | B. Ptet_RhlR (6A1) Prhl_GFP (3K3) #2<br> | |
− | + | C. Ptet_RhlR (6A1) Prhl(LR)_GFP (3K3) #1<br> | |
+ | D. Ptet_RhlR (6A1) Prhl(LR)_GFP (3K3) #2<br> | ||
+ | E. Ptet_RhlR (6A1) Prhl(RL)_GFP (3K3) #1<br> | ||
+ | F. Ptet_RhlR (6A1) Prhl(RL)_GFP (3K3) #2<br> | ||
+ | G. Ptet_RhlR (6A1) Prhl_a_GFP (3K3) #1<br> | ||
+ | H. Ptet_RhlR (6A1) Prhl_a_GFP (3K3) #2<br> | ||
+ | I. Ptet_RhlR (6A1) Placuv5_GFP (3K3) #1…positive control<br> | ||
+ | J. Ptet_RhlR (6A1) ΔP_GFP(3K3) #1…negative control<br> | ||
+ | |||
+ | [[Image:titech2013_parts_K1139150_exp_Fig1.jpg|frameless|none|250px||]] | ||
+ | |||
+ | <b>2. Assay protocol</b><br> | ||
+ | 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> | ||
+ | 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> | ||
+ | 3.Incubate the fresh cultures in 37°C until the observed OD590 reaches 0.3 (Actual value 0.42).<br> | ||
+ | 4.Add 30 microL of 500 microM C4HSL or DMSO as listed below:<br> | ||
+ | A-5 μM: A + C4HSL<br> | ||
+ | A-0 μM: A + DMSO<br> | ||
+ | B-5 μM: B + C4HSL<br> | ||
+ | B-0 μM: B + DMSO<br> | ||
+ | C-5 μM: C + C4HSL<br> | ||
+ | C-0 μM: C + DMSO<br> | ||
+ | D-5 μM: D + C4HSL<br> | ||
+ | D-0 μM: D + DMSO<br> | ||
+ | E-5 μM: E + C4HSL<br> | ||
+ | E-0 μM: E + DMSO<br> | ||
+ | F-5 μM: F + C4HSL<br> | ||
+ | F-0 μM: F + DMSO<br> | ||
+ | G-5 μM: G + C4HSL<br> | ||
+ | G-0 μM: G + DMSO<br> | ||
+ | H-5 μM: H + C4HSL<br> | ||
+ | H-0 μM: H + DMSO<br> | ||
+ | I-5 μM: H + C4HSL <br> | ||
+ | I-0 μM: I + DMSO<br> | ||
+ | J-5 μM: H + C4HSL <br> | ||
+ | J-0 μM: J + 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 9000 Xg, 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).<br> | ||
+ | |||
+ | |||
+ | ===Results=== | ||
+ | Fig. 1 shows the fluorescence intensity detected by flow cytometer.<br> | ||
+ | |||
+ | |||
+ | [[Image:titech2014_parts_K1529301_Fig2.jpg|thumb|center|310px|<b>Fig. 1.</b> Fluorescence intensity detected by flow cytometer]]<br> | ||
+ | |||
+ | <br> | ||
+ | |||
+ | 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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|}; | |}; | ||
<!-- End of the user review template --> | <!-- End of the user review template --> | ||
− | <!-- DON'T DELETE --><partinfo> | + | <!-- DON'T DELETE --><partinfo>BBa_K1139150 EndReviews</partinfo> |
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
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.
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
UNIQ03aee26aa88d6299-partinfo-00000001-QINU UNIQ03aee26aa88d6299-partinfo-00000002-QINU