Part:BBa_K1164002

LacI repressor tagged with yBFP

This is the LacI repressor from E.coli tagged with yeast codon optimized BFP. LacI will bind with high specificity to the the operator lacO and is inhibited in the presence of IPTG. BFP is a monomeric fluorescent protein that has an excitation peak of 399 nm and an emission peak of 465 nm. It was yeast codon optimized through DNA synthesis for the purpose of expressing in S. cerevisiae tagged to various repressors and activators.

This is an updated version of BBa_K642001 that is functional in S. cerevisiae.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1044
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 595
  
• 1000
  COMPATIBLE WITH RFC[1000]

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The above data was collected from the following yeast strain:
ade2:: Kanmx-rtact1-pgal-gfp
ade4:: Natmx – rtact1-pgal-laci-bfp
In the above construct, pgallx drives the production of GFP and is repressed by Laci. As shown in the graph above, the repressor, Laci (tagged with BFP), remains relatively constant over the entire IPTG gradient: a chemical that represses the activity of Laci. Consequently, as IPTG concentration increases, Laci inhibition should be increasingly inhibited, resulting in more GFP being transcribed, which is supported by the above data. The levels of fluorescence then stabilize and achieve steady state.

Reference

1. Ellis T, Wang X, Collins JJ. Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nat Biotechnol. 2009;27:46