Part:BBa_K1642009

Expression of GFP under the control of Pdark

Pdark-GFP is a plasmid used to test whether the promoter Pdark works as we expect.

Up and Down are sequences uesd for homologous recombination.

Figure1. Control mechanism of Pdark. (a) Under natural light, the green light component? induces phosphorylation of CcaR, and the binding of it to Pdark prevent the binding of RNA polymerase, thus inhibiting expression of halorhodopsin. (b) Under darkness, RNA Polymerase binds to Pdark and initiate the transcription of halorhodopsin as usual.

Pdark(BBa_K1026009) is a “dark-sensing” promoter combines PcpcG2 and a constitutive promoter from E.coli. Pdark contains binding site of CcaR in PcpcG2, which overlaps with the binding site of RNA polymerase. Therefore when phosphorylated CcaR binds to it, the binding of RNA polymerase to Pdark will be blocked and transcription can’t be initiated.

RBS is the RNA binding site, which will hopefully increase the expression level of GFP.

PcpcB is a constitutive promoter in Synechocystis sp. strain PCC, and Kana is the gene sequence for kanamycin resistance, so that bacteria which are successfully transferred this plasmid can be screened on the plate.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 611
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2995
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 481
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1294