Difference between revisions of "Part:BBa K1088009"

Revision as of 21:21, 25 September 2013

B. subtilis dxs-GFP protein fusion (lac promoter with LVA-tagged lac inhibitor (LacI:LVA) - IPTG ind

This part consist of the dxs gene derived from B. subtilis fused to GFP at the translational level with a 10 AA linker between the proteins. The reporter fusion is under the control of the lac promoter and has a strong RBS.

To repress expression from the lac promoter, the lacI:LVA (gene under a constitutive promoter, with a strong RBS and a efficient terminator) was placed counter-clockwise to the reporter fusion. The repression can be relieved with addition of IPTG, which binds and inhibits the function of LacI:LVA.

The levels of expression was meassured in E. coli K-12 MG1655 carrying the part using fluorescence activated cell sorting (FACS). The experiment proved that the reporter fusion was only very weakly expressed when the strain was grown without IPTG in the media, and that the reporter fusion was expressed at much higher levels after addition of IPTG. Though, in the same experiment we compared the LVA-tagged LacI with the natural and found that the natural LacI responded better to the IPTG addition. See experience for more details.

This Brick was build to test induction time and IPTG concentration for induction of a similar device (BBa_K1088013) which lacks the linker and GFP.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 2505
Illegal EcoRI site found at 3162
Illegal PstI site found at 2563
Illegal PstI site found at 3009
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 2505
Illegal EcoRI site found at 3162
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal PstI site found at 2563
Illegal PstI site found at 3009
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 2505
Illegal EcoRI site found at 3162
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 2505
Illegal EcoRI site found at 3162
Illegal PstI site found at 2563
Illegal PstI site found at 3009
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 2505
Illegal EcoRI site found at 3162
Illegal PstI site found at 2563
Illegal PstI site found at 3009
Illegal NgoMIV site found at 2462
Illegal AgeI site found at 2355
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 2304
Illegal BsaI.rc site found at 4018
Illegal SapI.rc site found at 3003

@@ Line 6: / Line 6: @@
 To repress expression from the lac promoter, the ''lacI:LVA'' (gene under a constitutive promoter, with a strong RBS and a efficient terminator) was placed counter-clockwise to the reporter fusion. The repression can be relieved with addition of IPTG, which binds and inhibits the function of LacI:LVA.
-The levels of expression was meassured in ''E. coli'' K-12 MG1655 using fluorescence activated cell sorting (FACS). The experiments proved that there was low expression when grown without IPTG and that there was high expression after addition of IPTG. Though, in the same experiment we compared the LVA-tagged LacI with the natural and found that the natural LacI responded better to the IPTG addition. See experience for more details.
+The levels of expression was meassured in ''E. coli'' K-12 MG1655 carrying the part using fluorescence activated cell sorting (FACS). The experiment proved that the reporter fusion was only very weakly expressed when the strain was grown without IPTG in the media, and that the reporter fusion was expressed at much higher levels after addition of IPTG. Though, in the same experiment we compared the LVA-tagged LacI with the natural and found that the natural LacI responded better to the IPTG addition. See experience for more details.
 This Brick was build to test induction time and IPTG concentration for induction of a similar device (BBa_K1088013) which lacks the linker and GFP.