Difference between revisions of "Part:BBa K2230016"
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Promoter Pcar [BBa_K861171] is a glucose responsive promoter created by WHU-China in 2012. Pcar promoter region was de novo  designed with overlapping of CRP and RNA polymerase binding site. The initiation of transcription by RNA polymerase may be hindered by the binding of CRP, which occurs at the formation of cAMP-CRP complex in the low concentration of glucose. In other words, when the amount of glucose is high enough, Pcar would be turned on after the leaving of CPR due to the low concentration of cAMP, and vice versa.  
 
Promoter Pcar [BBa_K861171] is a glucose responsive promoter created by WHU-China in 2012. Pcar promoter region was de novo  designed with overlapping of CRP and RNA polymerase binding site. The initiation of transcription by RNA polymerase may be hindered by the binding of CRP, which occurs at the formation of cAMP-CRP complex in the low concentration of glucose. In other words, when the amount of glucose is high enough, Pcar would be turned on after the leaving of CPR due to the low concentration of cAMP, and vice versa.  
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<span class='h3bb'>Sequence and Features</span>
 
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===Functional Parameters===
 
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Revision as of 15:58, 20 October 2017


Pcar-wRBS-PhlF-T-Pr-sRBS-GFP-sRBS-lysis/pSB1C3

Promoter Pcar [BBa_K861171] is a glucose responsive promoter created by WHU-China in 2012. Pcar promoter region was de novo designed with overlapping of CRP and RNA polymerase binding site. The initiation of transcription by RNA polymerase may be hindered by the binding of CRP, which occurs at the formation of cAMP-CRP complex in the low concentration of glucose. In other words, when the amount of glucose is high enough, Pcar would be turned on after the leaving of CPR due to the low concentration of cAMP, and vice versa.


PhlF repressor system contains the repressor PhlF [BBa_K1725041] and the PhlF repressible promoter [BBa_K1725001] created by Glasgow in 2015. PhlF could repress GFP fluorescence intensity by 83-fold according to the study of Glasgow’s work.


Lysis gene [BBa_K117000] created by NTU-Singapore in 2008 encodes Lysis protein which could not only lyse bacterial cell membrane but also activate the endonuclease of Colicin E7 (ColE7). The lysis-colicin is one class of bacteriocins which are produced to response to worsening environmental conditions and outcompete other bacteria1.


We’ve innovated this year a novel glucose responsive repressor system (Pcar-wRBS-PhlF-T-Pr-sRBS-GFP/pSB1C3 [BBa_K2230012]) by connecting these two system and extend the function of them. Furthermore, based on this new system, we assembled lysis and nuclease genes to the device and created the suicide circuit controlled by the presence of glucose (Pcar-wRBS-PhlF-T-Pr-sRBS-GFP-sRBS-lysis-sRBS-NucA/pSB1C3 [BBa_K2230017]).



Cloning: wRBS-lysis was amplified from Lysis gene (BBa_K117000) using a primer with weak RBS sequence (B0032) and assembled with Pcar-wRBS-PhlF-T-Pr-sRBS-GFP/pSB1C3 (BBa_K2230012)

Function: Suicide genes of lysis was driven under the PhlF-repressed promoter (Pr). And the expression of the repressor (PhlF) would be induced upon glucose by a glucose responsive promoter (Pcar). That is, the bacteria can be killed by the suicide genes in the absence of (or running out of) glucose.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1501