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Expression in different E.coli strains

Microbiology Molecular Biology

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#1 nvramakanth

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Posted 24 February 2014 - 05:43 AM

Hi,

 

I am working on a BAC plasmid. I have this plasmid in both E.coli DH5a and E.coli K12. What I find strange is , I don't see good expression in E.coli K12. Do anyone of you have encountered the same? If not what would be the possible option to make the plasmid work in E.coli K12. I have tried with different range of inductions. But nothing helped. Both the strains are endonuclease negative.

 

Thanks. Kindly help



#2 phage434

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Posted 24 February 2014 - 06:34 AM

K12 is the generic name for most species of lab E. coli. Your DH5a is a strain of K12. I don't know what your other strain is, but there are substantial differences between the strains, and without knowing more about the specific strain, little can be said. Some BACs have inducible copy number, so you could try adding IPTG to see if there was an effect. Why do you care?



#3 nvramakanth

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Posted 24 February 2014 - 07:08 AM

Let me be more specific:

 

I knew K12 and DH5a are variants of MG1655 strain. The problem actually is, reviewers these days started quoting that E.coli DH5a is a weaker strain when compared to K12 (For reasons unknown, which was very strange to us). So we decided, whatever the plasmids we construct should be  studied in K12. For cloning, screening the colonies and routine stuff we use E.coli Dh5a but for the studies we move the plasmid into K12.

 

Strain details:

 

E.coli K12: Can be found here. http://cgsc.biology....in.php?ID=64965

While DH5a is the routine one.

 

The BAC plasmid we use is a single copy F plasmid. This plasmid works pretty well with different concentrations of IPTG(10uM to 1mM) in DH5a but not in K12 which made me to post here. I need to develop an induction curve using this plasmid. Thats why I care smile.png  

 

Thanks. 



#4 phage434

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Posted 24 February 2014 - 07:54 AM

For starters, please use the strain name (BW25993) rather than "K12" to refer to this strain. You should compare the genotype of DH5a with BW25993 -- you can check the meaning of various genotype markers here:

http://openwetware.o..._coli_genotypes







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