Difference between revisions of "Part:BBa K2116025"

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Two copies of EsaR were placed under the control of individual constitutive promoters [Bba_J23118] and expressed on a medium-low copy plasmid (replication origin pBR322/rop). Based on the 2013 paper by Shong&Collins we decided to test AHL concentrations in the range of 0 to 10 000nM, since in all combinations of esabox placements tested in this paper showed saturation at 10 000nM as seen below:
 
Two copies of EsaR were placed under the control of individual constitutive promoters [Bba_J23118] and expressed on a medium-low copy plasmid (replication origin pBR322/rop). Based on the 2013 paper by Shong&Collins we decided to test AHL concentrations in the range of 0 to 10 000nM, since in all combinations of esabox placements tested in this paper showed saturation at 10 000nM as seen below:
  
[[File:BBa K2116025 Collinsdata.png|200px|frame|center|upright=0.2|Dose response curves of EsaR, tested on promoters with various esabox placements. (Shong&Collins 2013 [1]).]]
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[[File:BBa K2116025 Collinsdata.png|200px|thumb|center|upright=100|Dose response curves of EsaR, tested on promoters with various esabox placements. (Shong&Collins 2013 [1]).]]
 
   
 
   
 
We could not observe the same saturation behaviour in our tests,  
 
We could not observe the same saturation behaviour in our tests,  

Revision as of 15:02, 16 October 2016


EsaR repressible promoter

An esabox is an 18bp sequence to which the transcriptional regulator EsaR can bind. We placed one esabox right after the normally constitutive Anderson promoter [Bba_J23118] to create an EsaR repressible promoter. Transcription can be initiated by the specific AHL EsaR responds to [N-(3-oxo-hexanoyl)-L-homoserine lactone].

When characterising this part, we used the D91G variant of EsaR, which has been documented to be more responsive to lower AHL concentrations compared to the wild type EsaR [1]. This EsaR variant was obtained through addgene, and can be found on the registry [BBa_K2116001].

Two copies of EsaR were placed under the control of individual constitutive promoters [Bba_J23118] and expressed on a medium-low copy plasmid (replication origin pBR322/rop). Based on the 2013 paper by Shong&Collins we decided to test AHL concentrations in the range of 0 to 10 000nM, since in all combinations of esabox placements tested in this paper showed saturation at 10 000nM as seen below:

Dose response curves of EsaR, tested on promoters with various esabox placements. (Shong&Collins 2013 [1]).

We could not observe the same saturation behaviour in our tests,

Dose response curve of EsaR.



References: [1] Shong, Jasmine and Cynthia H. Collins. "Engineering The Esar Promoter For Tunable Quorum Sensing-Dependent Gene Expression". ACS Synth. Biol. 2.10 (2013): 568-575.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]