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How to insert genes into chromosome of E. coli


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#1 donny

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Posted 25 April 2012 - 11:05 AM

I am planning on inserting some genes into the chromosome of E. coli. How should I choose where to insert the genes? What are the easiest methods for gene insertion/replacement in E. coli? Some references will be nice. Thanks!

#2 donny

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Posted 26 April 2012 - 05:00 PM

Ok, I think I'll try adapting this method using a combination of RED and P1 transduction.
http://www.ncbi.nlm....pubmed/22127754

Still the question, where to insert? I will be generating foreign metaboites so there no way to know if non-essential genes, ie those in the KEIO collection, are actually important for the tolerance towards these metabolites. I'm thinking of inserting the genes into "barren" region in the genome. For example, as shown in the attached photo, there is a ~5kbp region that has apparently no genes. Is it safe to insert genes into this region?

e coli genome.jpg




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