I used codon optimized Plasmodium falciparum apicoplast helicase gene inserted into pet28a vector system (from Genscript) transformed into Rosetta 2DE3. I did transformation for 3-4 times 1-2 times I got one colony and another time I did not get any colony. What could be the reason ? I also checked my competent cell and I got result. Even When I transformed this plasmid inserted gene into DH5 alpha , I got good colony and from that I prep.plasmid and then did transformation into Rosetta2 DE3 that time I got good result. But directly from stock into Rosetta2DE3 I could not get anything why ?
Hello Jaishree,
I can see two possible explanations to your observations. First, it could be that your DH5 alpha competent cells are inherently more competent than the Rosetta2DE3 cells. This would be consistent with the higher number of colonies obtained with the DH5 alpha strain; once you prepare the plasmid and retransform Rosetta2 DE3, you probably use tens of nanograms of covalently closed plasmid DNA, which is optimal for efficient transformation. However, there is also a possibility that the poor efficiency of transformation in Rosetta2 DE3 is due to incomplete repression and leakage expression of the helicase gene, which may be detrimental for the host. If so, it may be a good idea to switch to a host that affords a more stringent control of expression, such as BL21AI (arabinose-inducible), in order to avoid selecting low- or non-producer segregants. As you mention that the gene has been codon-optimized (presumably for E. coli), you don't need a strain like Rosetta that provides rare tRNAs.
Hi.,
Transformation is the process by which an organism acquires exogenous DNA. Transformation can occur in two ways: natural transformation and artificial transformation. Natural transformation describes the uptake and incorporation of naked DNA from the cell's natural environment.
The two most popular methods of bacterial transformation are (1) heat shock of chemically prepared competent cells (chemical transformation), and (2) electroporation of electrocompetent cells.
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