Difference between revisions of "Part:BBa K3076213"

 
 
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<partinfo>BBa_K3076213 short</partinfo>
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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.
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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.
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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.
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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.
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3076213 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K3076213 parameters</partinfo>
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Latest revision as of 17:05, 8 October 2019


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

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.

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.

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.

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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 31
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]