[Microbiology] Please advise

mehru nisha via microbio%40net.bio.net (by nishamirsha2002 from yahoo.com)
Mon Nov 10 19:31:16 EST 2008

Dear sir,
Good day to you.
I'm Nisha from Malaysia. I'm a Master student working on live attenuated vaccine on Shigella flexneri 2a.
I have come across your article entittle "vector to knockout E. coli genes". I am working on mutating 2 genes in Shigella namely, apy and hemA gene. Currently im working on hemA gene. I have successfully mutate the gene by inserting a kannamycin marker and i have transformed the mutated gene in a suicidal vector called pWM91.
But, i unfortunately i have problem  in deleting the wild hemA gene in Shigella. I have transformed my construct into Shigella by conjugation with E.coli and i have obtained merodiploids. I have tried sucrose selection many many times, its already 5 months now-its still a failure!!!!
I'm getting worried and frustrated..
Can you please advise me sir?
Thank you for your kindness.

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