Part:BBa_K2230011

PI-wRBS-PhlF-T-Pr-sRBS-GFP/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.

PI promoter [BBa_K861170] is a modified version to Pcar with correction of -10 position in the promoter region, which gives the promoter stronger activity and a weaker CRP interaction. The PI promoter [BBa_K861170] was used as a major part in the work of team NCKU_Tainan in 2016. These two parts have been used in our work.

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.

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

Pr-sRBS-GFP (BBa_K1725001) with strong RBS (B0034) was assembled with PI-RBS-PhlF-T/pSB1C3 (BBa_K2230009)

Function

The expression of GFP driven by PhlF-repressed promoter (named Pr here) was dose-dependently regulated by PhlF repressor and reduced upon an increasing concentrations of glucose. In other words, the intensity of GFP increased when glucose gradually ran out.

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