What is the best (i.e. highest efficiency, fastest) genomic integration protocol for E. coli?

01 January 1970 1 4K Report

Hi all,

with the rise of CRISPR there has been a wave of genomic modification techniques.

In your opinion and judging from your experience what is the best (i.e. highest efficiency, fastest), protocol for integrating genes/expression cassettes (up to ~5kb) into the genome of E. coli?

I'm looking forward to your comments.

Cheers,

Michael

Hanna Alalam

Your best bet is lambda red coupled with CRISPR:

https://aem.asm.org/content/81/15/5103

Fragments larger that 2.5kb are very difficult to integrate using lambda red (and also a marker is required):

Article Site-specific chromosomal integration of large synthetic constructs

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