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help me in getting gene expression
Posted by: garima29 (IP Hidden, New member, 2)
Date: June 22, 2006 01:17AM
Hello everybody,
I want to discuss problem with you all. i have synthesized one gene and cloned it into pQE-80 vector. this gene has both ATG and TAA as start and stop codon. i have checked my clone with double digestion also. the problem is i am not getting expression. i have used SG13009 competent cell. will any other expression host will help me in geetting expression. please hepl meout. Garima
Re: help me in getting gene expression
Posted by: charan (IP Hidden, Junior member, 14)
Date: June 26, 2006 08:45AM
Well firstly I have some questions for you:
1. Did you sequence the entire gene? I assume you PCRed the gene from somewhere and cloned into your plasmid. When you do this, there are chances that you might have some additional mutations that might have introduced a stop codon or altered the reading frame. So I would sequence the gene first. 2. What is the genotype of SG 13009 cells? 3. what is the gene that you are cloning? I mean is the codon optimized for your expression systemi.e., assuming that SG 13009 is an E coli strain, are the codons in the gene that you cloned optimized for E coli expression? Some codons are not well transcribed in a heterogenous background. If everything looks good , you might want to introduce an additional Ribosome Binding Site in front of the start codon.
Re: help me in getting gene expression
Posted by: wufgang (IP Hidden, New member, 1)
Date: July 26, 2006 08:27PM
Rare codons might affect protein expression. Try codon optimization. There are many software applications for this purpose: [www.evolvingcode.net]
One program developed by our group is called "synthetic gene designer" (http://www.evolvingcode.net/codon/sgd/index.php)
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