Difference between revisions of "Part:BBa K4342009"
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<h1>Usage and Biology</h1> | <h1>Usage and Biology</h1> | ||
| − | [[File:AcrB_Deletion.png|200px|thumb|right|This gel demonstrates that the tdk/kan cassette replaced the | + | [[File:AcrB_Deletion.png|200px|thumb|right|This gel demonstrates that the <i> tdk/kan </i> cassette replaced the <i> acrB </i> gene and then was knocked out to produce a scarless deletion of <i> acrB </i> ]] |
| − | The | + | The <i> acrB </i> gene participates in the efflux of β-lactam antibiotics such as ampicillin and carbenicillin. The efflux process makes ADP1 more resistant to these antibiotics. Deleting the <i> acrB </i> gene increases ADP1's susceptibility to ampicillin, carbenicillin, and other β-lactams. Additionally, <i> acrB </i> gene is non-essential for ADP1’s survival, thus this gene serves as an ideal location for inserting genetic constructs. |
<h1>Design</h1> | <h1>Design</h1> | ||
| − | The acrB upstream homology part comprises the 1000 base pair region directly upstream of the acrB gene in ADP1. This part has bsaI and bsmbI restriction sites attached to the 3’ end which are designed to delete the acrB gene through a two-step process involving selection and counterselection. | + | The <i> acrB </i> upstream homology part comprises the 1000 base pair region directly upstream of the <i> acrB </i> gene in ADP1. This part has bsaI and bsmbI restriction sites attached to the 3’ end which are designed to delete the <i> acrB </i> gene 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 5’ end of the tdk/kan cassette [https://parts.igem.org/Part:BBa_K4342000 (BBa_K4342000)] creating the | + | The <b>bsaI site</b> is designed to ligate to the 5’ end of the <i> tdk/kan </i> cassette [https://parts.igem.org/Part:BBa_K4342000 (BBa_K4342000)] creating the <i> acrB 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 | + | The <b>bsmbI site</b> is designed to ligate to the 5’ end of the <i> acrB </i> downstream homology [https://parts.igem.org/Part:BBa_K4342010 (BBa_K4342010)] creating the <i> acrB rescue cassette </i> homologies composite part (BBa). This composite part permits the counterselection of transformants when plating on Azidothymidine (AZT). |
<h1>Characterization</h1> | <h1>Characterization</h1> | ||
<h1>References</h1> | <h1>References</h1> | ||
Revision as of 20:47, 6 October 2022
acrB Upstream
Contents
Usage and Biology
The acrB gene participates in the efflux of β-lactam antibiotics such as ampicillin and carbenicillin. The efflux process makes ADP1 more resistant to these antibiotics. Deleting the acrB gene increases ADP1's susceptibility to ampicillin, carbenicillin, and other β-lactams. Additionally, acrB gene is non-essential for ADP1’s survival, thus this gene serves as an ideal location for inserting genetic constructs.
Design
The acrB upstream homology part comprises the 1000 base pair region directly upstream of the acrB gene in ADP1. This part has bsaI and bsmbI restriction sites attached to the 3’ end which are designed to delete the acrB gene 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 acrB 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 acrB downstream homology (BBa_K4342010) creating the acrB rescue cassette homologies composite part (BBa). This composite part permits the counterselection of transformants when plating on Azidothymidine (AZT).