Part:BBa_K1744000:Design

araC-PBAD-vcrx028-ampR

Design Notes

The protein produced when the expression of vcrx028 is induced is a toxin, so you MUST repress this arabinose killswitch by adding glucose. Otherwise, you will get either no positive clones or highly mutated toxin that won't work well. In fact, the toxin sequence used is mutated from the native one, but proven good killing efficiency when induced. The part could not be BioBrick standardized for XbaI and PstI. It can still be digested using NotI or EcoRI + SpeI.

Sources

A RBS was selected through rational design and added with a primer to pVCR94's toxin coding sequence and it was cloned in the plasmid pBAD30 (using the site EcoRI). The part itself is a region of the resulting plasmid, from araC to bla.

References

Datsenko KA, Wanner BL, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc Natl Acad Sci USA 97, 6640(5), 2000.

Reddy TR, Kelsall EJ, Fevat LMS, Munson SE, Cowley SM, Differential Requirements of Singleplex and Multiplex Recombineering of Large DNA Constructs, PLoS ONE 10(5), 2015.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1619
  Illegal PstI site found at 1631
  Illegal PstI site found at 2708
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1227
  Illegal PstI site found at 1631
  Illegal PstI site found at 2708
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1166
  Illegal BamHI site found at 1613
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1619
  Illegal PstI site found at 1631
  Illegal PstI site found at 2708
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1619
  Illegal PstI site found at 1631
  Illegal PstI site found at 2708
  Illegal AgeI site found at 1001
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1815
  Illegal BsaI site found at 2882
  Illegal SapI site found at 983