Part:BBa_K4342011

pbpG Downstream Homology

Usage and Biology

This gel demonstrates that the tdk/kan cassette replaced the AcrB gene and then was knocked out to produce a scarless deletion of AcrB

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 upstream 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 3’ 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 5’ 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 downstream homology (BBa_K4342012) creating the ΔpbpG homologies composite part (BBa). This composite part permits the counterselection of transformants when plating on Azidothymidine (AZT).

References

This part was designed to ligate to the tdk/kan cassette and the ACIAD2049 Downstream Homology via bsaI and bsmbI digestions.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]