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Plasmid design

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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Plasmid design

Postby labfairy » Tue Dec 06, 2011 11:31 am

Hi,
I am trying to design a plasmid to be synthesised with two genes in the MCS, and appropriate RE's. I will then subclone this into a number of other vectors pET and one other.

The question I have is - do i have to include a specific sequence between the two genes as a transcription initiation of gene 2? Or can i go straight from the stop codon of gene 1 to the start codon of gene 2?

Thanks for your help!
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Postby JackBean » Tue Dec 06, 2011 1:52 pm

in what organism will you transcribe it? Bacteria (but probably every organism) should be able to do transcriptional fusion, so that two genes would be transcribed from one promoter, but the second gene would have lower expression. If you want the same level of expression, you should probably use two promoters.
http://www.biolib.cz/en/main/

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Postby labfairy » Wed Dec 07, 2011 5:22 pm

Thanks for your help - the plasmid will be going into bacterial system. I'm hoping that the level of expression won't be too different.
Thanks
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Postby canalon » Thu Dec 08, 2011 1:17 am

Or use a novagen Duet plasmid. They are designed for co-expression study and each gene has an independant initiation sequence, with two MCS and everything to make sure that it is easy to have your 2 proteins expressed at the same level.
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any proof. (Ashley Montague)
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