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Chloramphenicol resistance gene and Chemicomps E. coli (TOP10, Mach1)


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#1 ruiN

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Posted 18 June 2009 - 12:58 PM

I use the Cm resistance gene from L. lactis (gr+) as a selectable marker for my cloning, and it seems to make TOP-10 and Mach1 cells awfully sick. They're in fact resistant (gene is expressed) but they're just not growing well. Colonies take forever to grow and are tiny. Is it possible that the nature of the gene is not fully compatible with E. coli? Has anybody seen such a thing?

Thanks!

#2 Carlton H

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Posted 19 June 2009 - 09:19 AM

ruIN,

I think it's more likely that your Cm resistance gene is underexpressed and isn't conferring full resistance. Did you quantify your expression levels at all?

Cheers,
-Carlton
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#3 phage434

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Posted 19 June 2009 - 03:03 PM

If this is the cassette out of pC194, then it expresses poorly in E. coli, and is also inducible. The 5' UTR contains a stem loop which inhibits translation.

Horinouchi S, Weisblum B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol. 1982 May;150(2):815-25. PMID: 6950931

Removal of the stem-loop allows constitutive resistance, but still significantly lower resistance than the normal E. coli cat gene expression. See http://partsregistry...Part:BBa_J70304 and the design note page for the part.




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