Team:Tokyo Tech/Projects/Lux-Tet hybrid promoter/index.htm

From 2012.igem.org

(Difference between revisions)
(Materials & Method)
(Materials & Method)
Line 21: Line 21:
Sample:  
Sample:  
-
A) pLuxR-Ptrc-GFP (JM2300)
+
A) pLuxR-Ptrc-GFP (JM2.300)
-
B) pLuxR-ΔP-GFP (JM2300).... negative control
+
B) pLuxR-ΔP-GFP (JM2.300).... negative control
-
C) pLuxR-PLac-GFP (JM2300)… positive control
+
C) pLuxR-PLac-GFP (JM2.300)… positive control
(3)Strain
(3)Strain

Revision as of 08:41, 26 September 2012

bar

Tokyotechlogo2012.png

Lux-Tet hybrid promoter assay

Materials & Method

[Back to "Lux-Tet hybrid promoter assay"]

(1) construction

To characterize Lux-Tet hybrid promoter (BBa-K934024), we constructed Plux/tet-GFP (BBa-K934025) by ligating the Lux-Tet hybrid promoter (BBa-K934024) to the upstream of promoterless GFP generator (BBa-I13504).


(2)Samples

Sample:

A) pLuxR-Ptrc-GFP (JM2.300)

B) pLuxR-ΔP-GFP (JM2.300).... negative control

C) pLuxR-PLac-GFP (JM2.300)… positive control

(3)Strain

JM2.300

(4)protocol

Sample:

A) pLuxR-Ptrc-GFP (JM2.300)

B) pLuxR-ΔP-GFP (JM2.300).... negative control

C) pLuxR-PLac-GFP (JM2.300)… positive control

Method:

1 ,Prepare overnight culture at 37℃ for 12hours.

2, Take 30μl of the overnight culture of samples into LB(3ml) + antibiotics (Amp 50μg/ml).(→fresh culture)

3,Dilute the flesh culture in 1:50 by the following conditions:

a) LB

b) LB + anhydrotetracycline (500ng/ ml)

c) LB + acylated homoserine lactone(1μM )

d) LB + anhydrotetracycline (500ng/ ml) + acylated homoserine lactone(1μM )

4, Incubate the flesh culture of diluted inducer cells for 2 hours.

5, Flow cytometer measurements for GFP expression of reporter cell.


[Back to "Lux-Tet hybrid promoter assay"]