Difference between revisions of "Part:BBa K3076300"

Revision as of 13:14, 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.