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Part:BBa_K1088009

Designed by: Andreas Kjær   Group: iGEM13_SDU-Denmark   (2013-08-20)
Revision as of 17:39, 25 September 2013 by P.R.A (Talk | contribs)

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 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.

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


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