Part:BBa_K389014:Design

VirA/G screening system test

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 647
  Illegal NheI site found at 2581
  Illegal NheI site found at 2604
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1632
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1768
  

Design Notes

• The virA gene is under the control of a medium strong constitutive promoter so there is enough VirA receptor expressed but not too much so the cell suffers from the expression (VirA is a membrane protein)
• The virG gene is mutated so it works without an rpoA subunit from Agrobacterium tumefaciens (YC Jung et al., 2004)
• double terminator (forward) to keep expression of kanamycin resistance by the constitutive promoter low
  • double terminator BBa_B0017 instead of BBa_B0015 because of problems mentioned with BBa_B0012
• only for testing the screening principle of mutated virA screening

Source

• virA from A. tumefaciens C58 (BBa_K389001)
• virG gene synthesized by Mr. Gene (BBa_K389002)
• vir promoter from A. tumefaciens C58 (BBa_K389003)
• kanR gene made from BioBrick BBa_P1003
• constitutive promoter, RBS and double terminator from parts.igem (BBa_J23110, BBa_B0034, BBa_B0017)

References

YC Jung et al. (2004) Mutants of Agrobacterium tumefaciens virG Gene That Activate Transcription of vir Promoter in Escherichia coli, Current Microbiol 49:334-340.