I am expressing few cellulolytic genes using few expression plasmids (Shuttle vectors).
My question is if instead of maintaining your heterologous gene in plasmid, if u use an integration vector that will transfer the entire cassette (carrying RBS promoter Signal peptide gene) into the host system (bacillus subtilis), does it improves the secretion of the gene more efficiently when compared to expression obtained when transformed as a plasmid.
Kindly share your experiences or articles proving integration vectors more efficient over expression plasmids. (in particular with Bacillus system)
This is a complicated question. Expression, when using the same expression signals, should be similar (though one could expect that a slight increase in copy number of a low copy number plasmid compared to single copy genomic might raise it a little). But, the stability of the - double crossover - genomic construct is much better, especially if you are producing harmful or toxic proteins (NB: these can be proteins that in itself are not toxic, but may become toxic at non-native levels). Plasmids more rapidly accumulate mutations or could be lost. If you, in the long run, are interested in curing the strain of the expression construct, this is ofcourse easier using the plasmid based system. Single crossover genomic constructs are somewhat in between these two; they can more rapidly be lost, but may be less leaky (due to single copy number) than plasmids.
I believe that integration of genes is better. It leads to better stability and efficiency. I worked with Lactococcus and observed that the mRNAs of a particular gene expressed in plasmid was unstable and had a very short half life. When the same genes were expressed after integration in genome the stability of the genes improved, improving product formation.
I don't really think chromosomal integration will necessarily give you higher expression, because you can have tens of copies of the target gene in plasmid. However, if you want to do industrial production, it is critical to integrate the cassette in the chromosome because this gives you much better genetic stability.
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