Difference between revisions of "Part:BBa K3076300"
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<partinfo>BBa_K3076300 short</partinfo>
 
<partinfo>BBa_K3076300 short</partinfo>
  
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==Description==
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<p><br>This part is designed to use as double-stranded (ds) DNA substrate for knocking out CopA gene in E. coli by Lambda Red combineering system.</br></p>
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<p>This part is designed to use as double-stranded (ds) DNA substrate for knocking out <i>CopA</i> gene in <i>E. coli</i> by Lambda Red recombineering system.</p>
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<p><br>The fragment contains homology sequences of 50 bp flanking a double terminator and a kanamycin resistance gene. The recombination site is at the 76 bp to 156 bp of the CopA gene. If the recombination succeeded, the kanamycin resistance gene will be inserted in between the CopA gene and disrupting CopA’s expression. Meanwhile, the kanamycin resistance gene can be used as a selection marker for successful recombination. A double terminator (BBa_B0015) was added at the 5’ end of the fragment to ensure the termination of CopA gene expression.</br><p/>
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<p><br>In order to use this substrate, simply amplifying this part by PCR from a plasmid. The PCR product is ready to be transformed directly for recombineering. The E. coli strain used for recombination should express lambda red recombineering genes, such as strain DY380. Otherwise, the lambda red recombineering system can be introduced to the E. coli strain by transforming plasmids with lambda red genes, such as pKD46.</br></p>
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<p><br>Due to the time constraint, we obtained the already knockout strain from Keio knockout strain library directly to carry out the functional study. This dsDNA substrate has not been tested yet.</br></p>
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<h3>Assay : </h3>
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<h4>Abstract of experiment</h4>
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<br><p><u>Graph 1: </u></p>
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<p><u>Graph 1:</u></p>
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==Usage and Biology==
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<p>This part contains homology sequences of 50 bp flanking a double terminator and a kanamycin resistance gene. The recombination site is at the 76 bp to 156 bp of the <i>CopA</i> gene. If the recombination succeeded, the kanamycin resistance gene will be inserted in between the <i>CopA</i> gene and disrupting the expression. Meanwhile, the kanamycin resistance gene can be used as a selection marker for successful recombination. A double terminator <html>(<a href="https://parts.igem.org/Part:BBa_B0015">BBa B0015</a>)</html> was added at the 5’ end of this fragment to ensure the termination of gene expression.</p>
  
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<h3>Results</h3>
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<p>To use this substrate, simply amplify this part by PCR. The PCR product is ready to be transformed and recombined. The <i>E. coli</i> strain used should express lambda red recombineering genes. On the other hand, the lambda red recombineering system can be introduced to the <i>E. coli</i> strain by transforming the <i>E. coli</i> with plasmids containing lambda red genes, such as pKD46.</p>
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</br>
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<h3>Future work</h3>
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<p>Due to the time constraint, we obtained the knockout strain from Keio knockout strain library directly to carry out the functional study. This dsDNA substrate has not been tested yet.</p>
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Revision as of 13:11, 11 October 2019

dsDNA substrate with KanR gene for CopA knockout in E. coli by Lambda Red Recombineering

Description

This part is designed to use as double-stranded (ds) DNA substrate for knocking out CopA gene in E. coli by Lambda Red recombineering system.

Usage and Biology

This part contains homology sequences of 50 bp flanking a double terminator and a kanamycin resistance gene. The recombination site is at the 76 bp to 156 bp of the CopA gene. If the recombination succeeded, the kanamycin resistance gene will be inserted in between the CopA gene and disrupting the expression. Meanwhile, the kanamycin resistance gene can be used as a selection marker for successful recombination. A double terminator (BBa B0015) was added at the 5’ end of this fragment to ensure the termination of gene expression.

To use this substrate, simply amplify this part by PCR. The PCR product is ready to be transformed and recombined. The E. coli strain used should express lambda red recombineering genes. On the other hand, the lambda red recombineering system can be introduced to the E. coli strain by transforming the E. coli with plasmids containing lambda red genes, such as pKD46.

Due to the time constraint, we obtained the knockout strain from Keio knockout strain library directly to carry out the functional study. This dsDNA substrate has not been tested yet.