Part:BBa_K3605010

Modified RFP generator

Since the existing part J04450, a RFP generator, has some leaking expression. We created this modified RFP generator,which only expresses RFP in the presence of IPTG.

Overview:

BBa_J04450 is a RFP generator driven by Plac promoter, which is regulated by LacI and induced by IPGT. Plac is one of the most common promoter in life science research field. It is mainly composed of Lac operon containing LacO site. LacI repressor, encoded by LacI gene, can bind to LacO site, inhibiting the binding of RNA polymerase to the promoter. So the expression of downstream genes is blocked. Serving as inducer, IPTG can bind with LacI repressor, making the latter’s conformation changes, so LacI is detached from LacO site, which enables the transcription of downstream genes. BBa_J04450 is a RFP generator driven by Plac promoter, however there is no LacI gene in it. Although the E. coli could express some LacI, it is not enough for inhibition RFP expression. So this RFP generator has some leakage expression, like the designer stated that some RFP express even without IPTG inducer presence.

This year, we constructed a new RFP generator (BBa_K3605010) also driven by Plac promoter. It contains LacI gene, which can lower significantly the RFP leakage expression. Both LacI and RFP are linked to the downstream of Plac, which is regulated by both LacI repressor and IPTG inducer. Using RFP as a reporter, its fluorescence intensity appears a lower leakage expression. This new RFP generator could be self-regulated because LacI protein can inhibit its self-expression, so that no excessive LacI expression which is considered as waste of resources. We detected the response of this generator to different concentration of IPTG, indicating that it could be inhibited by LacI, and induced well by IPTG.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1373
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1940
  Illegal AgeI site found at 2052
  
• 1000
  COMPATIBLE WITH RFC[1000]