Part:BBa_K2116025

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 Part: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 Part: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 observed EsaR response to the same AHL, but did not see the same saturation behaviour.

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]