Revision as of 17:46, 5 October 2022

pbpG 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 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).

Characterization

References

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]

@@ Line 4: / Line 4: @@
-<h1>Usage and Biology</h1>
+ <h1>Usage and Biology</h1>
+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.
 <h1>Design</h1>
+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 <b>bsaI site</b> is designed to ligate to the 5’ end of the tdk/kan cassette [https://parts.igem.org/Part:BBa_K4342000 (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 <b>bsmbI site</b> is designed to ligate to the 5’ end of the pbpG downstream homology [https://parts.igem.org/Part:BBa_K4342010 (BBa_K4342012)] creating the ΔpbpG homologies composite part (BBa). This composite part permits the counterselection of transformants when plating on Azidothymidine (AZT).
 <h1>Characterization</h1>
 <h1>References</h1>