Difference between revisions of "Part:BBa K1088009"
(Further explanation + description of functionality)
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<partinfo>BBa_K1088009 short</partinfo>
 
<partinfo>BBa_K1088009 short</partinfo>
  
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This is the same part as BBa_K1088008, with the exception of LacI under the control of a constitutive promoter to ensure regulation of the Lac Promoter
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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.
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This part consists of 9 subparts:
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A constitutive promoter: BBa_J23106
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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.
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A strong RBS: BBa_B0030
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The LacI repressor protein, regulating the lac promoter: BBa_C0012
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The levels of expression was meassured in ''E. coli'' K-12 MG1655 using fluorescence activated cell sorting (FACS). The experiments proved that their was low expression when grown without IPTG and that there, over longer time compared to natural LacI, was expression after addition of IPTG. See experience for more details.
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An artificial terminator: BBa_B1002
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and the rest is similar to BBa_K1088008
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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.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 19:04, 19 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 using fluorescence activated cell sorting (FACS). The experiments proved that their was low expression when grown without IPTG and that there, over longer time compared to natural LacI, was expression after addition of IPTG. 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