Part:BBa_K615000:Design

E. coli strain for His3-URA3 one-hybrid selection system

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 632
  Illegal PstI site found at 952
  Illegal PstI site found at 1762
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 632
  Illegal PstI site found at 952
  Illegal PstI site found at 1762
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 845
  Illegal BglII site found at 965
  Illegal BglII site found at 1478
  Illegal BglII site found at 1538
  Illegal BamHI site found at 1768
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 632
  Illegal PstI site found at 952
  Illegal PstI site found at 1762
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 632
  Illegal PstI site found at 952
  Illegal PstI site found at 1762
  Illegal NgoMIV site found at 181
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2458
  Illegal SapI site found at 121
  Illegal SapI site found at 331
  Illegal SapI.rc site found at 2305
  

Design Notes

This selection strain was adapted from the one-hybrid system presented by Meng et al (2005), but instead of having the zinc finger binding site and associated genes on a plasmid, we placed the selection construct directly onto the genome, thus ensuring that the system will not be lost in subsequent generations and that each cell has exactly one copy.

Genotype:

• HisB KO: 6 nucleotides (694-699) coding for amino acids V and E were deleted using MAGE.
• PyrF KO: Two early in-frame stop codons were made using MAGE to substitute 8T-->A and 16C-->G
• rpoZ KO: Lambda red was used to replace gene with Zeocin resistance cassette.
• Kanamycin resistance cassette—Zif268 binding site and weak promoter—His3—URA3: inserted into 1529620 genomic locus using lambda red.
• MutS KO: replaced by chloramphenicol resistance cassette.

Source

• Selection system was adapted from Meng et al (2005).
• Base strain was EcNR2, a modified E. coli strain as described in Wang et al (2009).
• URA3 and His3 genes were synthesized.

References

Harris H. Wang, Farren J. Isaacs, Peter A. Carr, Zachary Z. Sun, George Xu, Craig R. Forest, George M. Church. Programming cells by multiplex genome engineering and accelerated evolution. (2009). Nature, 460(7257):894-8.

Xiangdong Meng, Michael H Brodsky, Scot A Wolfe. A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors. (2005). Nature Biotechnology, 23(8): 988-994.