Dear all,
Recently I cloned two genes from streptobacillus moniliformis and Erysipelothrix rhusiopathiae into pGEM-T vector, and they were confirmed by sequencing results.
Then I tried to clone into constitutive expression vector, but after trying several times, still didn't work out. Double digestion and ligation sould be no problem, because the third gene from Anaerococcus prevotii was successfully inserted into this constitutive vector in the same batch expriment.
So now i am wondering is it possible because these are pathgenic bacteria, which will be toxic to E.coli when constitutive expressing?
I am really tired with molecular cloning. Any suggestions are welcome, and will help me.
Thanks in advance!
Biocat
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Doubt with recombinant expression genes from streptobacillus moniliformis and Er
Started by Biocat, May 27 2010 11:37 AM
2 replies to this topic
#2
Biocat
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Posted 30 May 2010 - 04:08 AM
I need help!
Biocat, on May 27 2010, 09:37 PM, said:
Dear all,
Recently I cloned two genes from streptobacillus moniliformis and Erysipelothrix rhusiopathiae into pGEM-T vector, and they were confirmed by sequencing results.
Then I tried to clone into constitutive expression vector, but after trying several times, still didn't work out. Double digestion and ligation sould be no problem, because the third gene from Anaerococcus prevotii was successfully inserted into this constitutive vector in the same batch expriment.
So now i am wondering is it possible because these are pathgenic bacteria, which will be toxic to E.coli when constitutive expressing?
I am really tired with molecular cloning. Any suggestions are welcome, and will help me.
Thanks in advance!
Biocat
Recently I cloned two genes from streptobacillus moniliformis and Erysipelothrix rhusiopathiae into pGEM-T vector, and they were confirmed by sequencing results.
Then I tried to clone into constitutive expression vector, but after trying several times, still didn't work out. Double digestion and ligation sould be no problem, because the third gene from Anaerococcus prevotii was successfully inserted into this constitutive vector in the same batch expriment.
So now i am wondering is it possible because these are pathgenic bacteria, which will be toxic to E.coli when constitutive expressing?
I am really tired with molecular cloning. Any suggestions are welcome, and will help me.
Thanks in advance!
Biocat
#3
pDNA
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Posted 30 May 2010 - 11:20 AM
interference of the sequences in e. coli is for sure possible ...what we experienced is that it is sometimes a real pain to clone prokaryotic sequences in prokaryotes.
Maybe you can give some details on the function of the proteins you try to clone?
I would not use a constituitive promoter for that task ...and instead use a promoter that is very leak proof (e.g. the arabinose inducible BAD-promoter or a pL lambda promoter, inducible by heat shock).
This gives you more control over your experiment ...and you can try to select clones under fully suppressed conditions ...maybe more successful than working with a constituitive promoter!
Good luck!
Regards,
p
Maybe you can give some details on the function of the proteins you try to clone?
I would not use a constituitive promoter for that task ...and instead use a promoter that is very leak proof (e.g. the arabinose inducible BAD-promoter or a pL lambda promoter, inducible by heat shock).
This gives you more control over your experiment ...and you can try to select clones under fully suppressed conditions ...maybe more successful than working with a constituitive promoter!
Good luck!
Regards,
p
