I am willing to design a suitable construct of two genes in a single reading frame, both of which could express with same efficiency under the regulation of a common promoter and RBS. I have to express that construct in bacteria, already i have pET29 A (+) expression vector. My problem is the termination codon of first gene, and initiation codon of second gene; how to manipulate these two codons that I will get such a polycistronic RNA from which two individual proteins can be produced. If anyone have such research papers, or views, please send me and give your opinions.
Thanks.
You put another ribosome binding before the second ORF. I doubt you will have same expression level of both genes though; my recommendation is to use two similar vectors with different selection markers only (e.g. same backbones with different antibiotic resistance), and put the two genes under identical promoter, and select with 2 drugs. You can test the expression levels with qPCR and western blot.
Dear Ian Hu,
Thanks,
I think, adding new RBS before second ORF will surely reduce its expression levels, however, first gene will express with normal efficiency. In your secnd suggesion, If, I will use similar vector with another selection marker only, and that having same backbone will not be taken up by bacterial cell due to plasmid compatibility problems. Tell me one thing, may I use such a construct
Promoter1+RBS1+MCS1+transcriptional terminator1+---------Promoter1 +RBS1
+MCS2+transcriptional terminator1+Ori Site+Marker gene
By doing so, I can express both genes with similar efficiency, and problem of using two vectors and two antibiotics will be sort out.
For your reference, compatibility may be higher than you think if you're not expressing them for too long:
http://peds.oxfordjournals.org/content/20/7/309.full
But yes your second scheme should work as well as long as the strain lacks recombinase (which is true for almost all cloning strains but not necessarily for expressing strains, mind this). I do assume however making this construct will bring you quite some trouble.
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