Difference between revisions of "Part:BBa K4342012"
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<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. | + | The <i> pbpG </i> gene codes for a penicillin-binding protein that increases ADP1’s resistance to β-lactam antibiotics. Thus deleting the <i> pbpG </i> gene makes ADP1 more susceptible to ampicillin, carbenicillin, and other β-lactams. Additionally, the <i> pbpG </i> gene is non-essential for ADP1’s survival and serves as an ideal location for inserting genetic constructs. |
<h1>Design</h1> | <h1>Design</h1> | ||
− | 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. | + | The <i> pbpG </i> downstream homology part comprises the 1019 base pair region directly upstream of the <i> pbpG </i> gene in ADP1. This part has bsaI and bsmbI restriction sites attached to the 5’ end which are designed to delete <i> pbpG </i> through a two-step process involving selection and counterselection. |
===BsaI Restriction Site=== | ===BsaI Restriction Site=== | ||
− | The <b>bsaI site</b> is designed to ligate to the 3’ 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. | + | The <b>bsaI site</b> is designed to ligate to the 3’ end of the <i> tdk/kan </i> cassette [https://parts.igem.org/Part:BBa_K4342000 (BBa_K4342000)] creating the <i> pbpG tdk/kan </i> cassette composite part (BBa). This composite part permits the selection of transformants through kanamycin resistance. |
===Bsmbi Restriction Site=== | ===Bsmbi Restriction Site=== | ||
− | The <b>bsmbI site</b> is designed to ligate to the 5’ end of the pbpG upstream homology [https://parts.igem.org/Part:BBa_K4342011 (BBa_K4342011)] creating the | + | The <b>bsmbI site</b> is designed to ligate to the 5’ end of the <i> pbpG </i> upstream homology [https://parts.igem.org/Part:BBa_K4342011 (BBa_K4342011)] creating the <i> pbpG </i> rescue cassette composite part (BBa). This composite part permits the counterselection of transformants when plating on Azidothymidine (AZT). |
<h1>Characterization</h1> | <h1>Characterization</h1> |
Revision as of 20:55, 6 October 2022
pbpG Downstream Homology
Contents
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 pbpG rescue cassette composite part (BBa). This composite part permits the counterselection of transformants when plating on Azidothymidine (AZT).
Characterization
References
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 300
Illegal PstI site found at 244 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 244
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 300
Illegal PstI site found at 244 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 300
Illegal PstI site found at 244 - 1000COMPATIBLE WITH RFC[1000]