Part:BBa_K4242013

Pci+RBS+fleQ+TT+ Pci+Ppel+RBS+gfp+TT(pHYD-3)

Uses factor PcI (BBa_R0051), strong RBS(BBa_J34801), protein FleQ coding region(BBa_K4242000), TT(BBa_B0015), PcI(BBa_R0051) and Ppel(BBa_K4242001), TT(BBa_B0015), RBS(BBa_J34801), gfp coding region(BBa_E0040), and TT(BBa_B0015). This part is a device to construct the expression of protein FleQ in order to repress the expression of downstream gene by binding to Ppel. FleQ is a transcription factor from P. aeruginosa which can bind to the promotor Ppel and inhibit the gene expression. However, this inhibition can be released when c-di-GMP binds to the FleQ and changes its spatial structure.

Usage and Biology

To test whether FleQ can repress gfp expression under the control of the tandem promoter, we cloned fleQ gene from the genome of P. aeruginosa and inserted it into pHYD-2, generating the plasmid pHYD-3 that contains the complete c-di-GMP biosensor. FleQ represses the transcription of gfp though inhibition of the RNA polymerase to access DNA by binding to Ppel when the level of c-di-GMP is low. At high intracellular c-di-GMP, FleQ undergoes a conformational change when it binds c-di-GMP, which relieves FleQ from pel promoter and leads to gfp gene expression under the control of the tandem promoter. As FleQ represses the expression of gfp by binding to the promoter Ppel, the fluorescence of E. coli/pHYD-3 and S. oneidensis/pHYD-3 expressing the repressor FleQ was significantly decreased compared to that of the strains with pHYD-2 that has no FleQ. Taken together, the results showed the tandem promoter is effective in promoting expression of the reporter gene gfp, and the transcription factor FleQ is able to decrease gfp expression level.

pHYD-3

fig.1 a|engineering vectors b| Fluorescence intensity in E.coli BL21 c| Fluorescence intensity in S. oneidensis MR-1

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 943
  Illegal XhoI site found at 1396
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 336
  Illegal NgoMIV site found at 1344
  Illegal NgoMIV site found at 1356
  Illegal NgoMIV site found at 1432
  Illegal NgoMIV site found at 1517
  Illegal AgeI site found at 745
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2567
  Illegal SapI.rc site found at 358
  Illegal SapI.rc site found at 1291
  Illegal SapI.rc site found at 1300