I want to express two mammalian gene into single plasmid of mycobacterium. In which way should these gene products should get secreted and separated?
So which kind of strategy I should follow and suitable plasmid to clone these genes.
Firstly, you have to fuse both genes through SOE PCR strategy. SOE is nothing, just a modified form of PCR principle which usually is being used for fusion or deletion of genes. during designing primer, put secretory protein signal sequence to your first protein and between two genes, introduce one cleavage site so that either you manually can cut both of expressed proteins or go through literature and find out what are those cutting site which can be cut by cellular machinery automatically at the time of expression.
Hi Sudeep, here are a few basics that need to be considered when expressing mammalian genes in bacteria. I am sure you are aware of them but it does no harm to restate them here.
a) Mammalian genes in bacteria need to have a prokaryotic promoter upstream of them (not a mammalian promoter)
b) Depending on the gene sequence you may get lower expression in bacteria if the mammalian sequence contains codons that are rare in bacteria (bacteria have a different codon usage). There is an interesting link here:
http://openwetware.org/wiki/CH391L/S12/TranslationRBSandCodons
You mention that you want the gene-products secreted so I assume it is not essential that they are in the same bacteria? If (for some reason) you want the genes in the same bacteria it might be best/easiest to transfect the bacteria with two separate plasmids (with different antibiotic resistances i.e. plasmid 1 = gene X Kano selection, plasmid 2 = gene Y Amp).
With the genes on separate plasmids there will be no need to run the genes off the same promoter (i.e. IRES/RBS).
Hope this helps,
Gary
Complete genome sequencing of M.tb revealed the existence of unique family of protein, PE/PPE, found exclusively in the genus Mycobacterium. These genes are so
named due to the presence of Pro-Glu (PE) and Pro-Pro-Glu (PPE) signature motifs near the N-terminus of their gene products
http://web.iitd.ac.in/~amittal/2012_SEH_NAR.pdf
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