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Point mutations in synthesis order of a 2.4 kb nucleotide fragment

Gene Synthesis

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3 replies to this topic

#1 IPI

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Posted 10 May 2013 - 08:50 PM

Hi,


I have ordered a 2400-bp nucleotide fragment (codon-optimized for Lactococcus lactis), to be synthesized and cloned in E. coli by a commercial firm. The company has now informed me that they were unable to produce the fragment in correct ORF, due to formation of a few disruptive point mutations. I was wondering:


1- Are these mutations happening because the sequence is toxic to E. coli?

2- How can I circumvent this problem?

3- Can removing the area where these point mutations have happened, solve this problem?


With many thanks in advance for your inputs,


IPI


Edited by IPI, 10 May 2013 - 08:52 PM.


#2 phage434

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Posted 13 May 2013 - 04:41 AM

Usually this means that the E. coli is reacting badly to the presence of the sequence. This could be expression of a toxic gene, or accidental creation of a plasmid origin. You could synthesize your gene paying careful attention to elimination of any promoters, and possibly by insertion of terminators. You might find it necessary to assemble and transform the DNA in a final step prior to insertion into L. lactis. If there are only a few mutations in a single place, PCR might be used to create a dsDNA fragment with correct sequence, which could then be transformed.
Or you could try a second synthesis provider. DNA 2.0 is pretty good when others fail.

#3 pDNA

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Posted 19 May 2013 - 05:01 AM

maybe you could make use of direct cloning approaches (i don't know if this also works for Lactococcus):
http://www.ncbi.nlm....pubmed/23098256

avoiding the use of E. coli for gram+ associated sequences can prevent a lot of troubles.

Best,
p

#4 Trof

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Posted 20 May 2013 - 10:39 AM

You could synthesize it in two parts (with overlaping fragments) in E. coli, or use a modular system of artificially synthetized smaller fragments, that would be complementary (now I just forgot the exact name of this type of cloning stategy, even if you killed me..)
Something like this: A modular assembly cloning technique, for example.



But if the sequence is indeed toxic to E.coli, and you want to use it in E.coli, you will probably face the same problem (but as I understood, you want it for Lactococcus, so maybe this is not an issue).

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