Part:BBa_K1642011

Expression of Halorhodopsin under the control of Pdrak

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

Pdark-HR is a plasmid used to test whether the protein halorhodopsin under the control of P dark works as we expect.

Up and Down are sequences used for homologous recombination.

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 HR.

Halorhodopsin (HR) proteins are light-driven inward-directed chloride pumps from halobacteria. We use this as our biodesalination driver which confers cyanobacteria the ability to absorb chloride to a significant degree.

Terminator is the transcription terminator for the E.coli RNA polymerase.

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 3215
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 481
  Illegal NgoMIV site found at 783
  Illegal NgoMIV site found at 942
  Illegal NgoMIV site found at 1263
  Illegal AgeI site found at 1134
  
• 1000
  COMPATIBLE WITH RFC[1000]