Part:BBa_K3076600

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

This part is designed to use as double-stranded (ds) DNA substrate for knocking out cutA 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 8 bp to 88 bp of the cutA gene. If the recombination succeeded, the kanamycin resistance gene will be inserted in between the cutA gene and disrupting cutA’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 cutA 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.

Assay :

Abstract of experiment

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Graph 1:

Graph 1:

Results

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