Part:BBa_K4342012

recJ Upstream

Usage and Biology

The pbpG gene codes for a penicillin-binding protein that increases ADP1’s resistance to β-lactam antibiotics. Thus deleting the pbpG gene makes ADP1 more susceptible to ampicillin, carbenicillin, and other β-lactams. Additionally, the pbpG gene is non-essential for ADP1’s survival and serves as an ideal location for inserting genetic constructs.

Design

The pbpG downstream homology part comprises the 1019 base pair region directly upstream of the pbpG gene in ADP1. This part has bsaI and bsmbI restriction sites attached to the 5’ end which are designed to delete pbpG through a two-step process involving selection and counterselection.

BsaI Restriction Site

The bsaI site is designed to ligate to the 3’ end of the tdk/kan cassette (BBa_K4342000) creating the pbpG tdk/kan cassette composite part (BBa). This composite part permits the selection of transformants through kanamycin resistance.

Bsmbi Restriction Site

The bsmbI site is designed to ligate to the 5’ end of the pbpG upstream homology (BBa_K4342011) creating the ΔpbpG homologies composite part (BBa). This composite part permits the counterselection of transformants when plating on Azidothymidine (AZT).

References

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 300
  Illegal PstI site found at 244
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 244
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 300
  Illegal PstI site found at 244
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 300
  Illegal PstI site found at 244
  
• 1000
  COMPATIBLE WITH RFC[1000]