I have a WT and null cell for a particular protein. I also have complemented set of cells which can be selected using kanamycin. For experimental issues I cannot use kanamycin. Will the plasmid be retained if the cells are grown and sub cultured multiple times without kanamycin? How long can it take to lose the plasmid without the selection pressure?
Lets do the math:
say you have a low copy plasmid; in order to cure one cell will need to end up with 0 plasmid copies upon segregation. If it ends up with 1 the plasmid will replicate again. Say its 50% chance for one plasmid to segregate. Then if theres 10 copies the chance of 0 is 1/1000.
Then you calculate the time it will overtake the culture with the fitness burden.If you express something, usually it will occupy a large percent of protein, say 10% synthesis resources. In that case expression plasmid will have a burden. For blank plasmid fitness burden should be very low. Plasmid is only a few kb. Genome is a few Mb. As long as you have >=1 plasmid, you cant cure, So gene synthesis burden is like
Dear Tias, I have read your question carefully and I hope I understand the experimental situation you face. If you have a plasmid (expressing a gene of interest) conferring kanamycin resistance, and have a host knock-out which also has a kanamycin marker, then I think it would be foolish to try to undertake experiments with both, as you will never be sure if the plasmid is present or not. Furthermore, unless you had some other marker on the plasmid you could select for (e.g. another antibiotic resistance gene) or screen for (e.g. b-galactosidase activity resulting from the expression of a fusion protein, PCR confirmation of the presence of the plasmid sequence, etc.), you could never be confident that you have managed to transform your host with the plasmid and that the plasmid is retained. I would suggest that the best way forward would be to either transfer the gene of interest from your plasmid into another which has a more suitable marker for selection, or to try an add a second antibiotic marker to your plasmid, most probably downstream of the gene of interest. Although Abel's comments above are valid, relying on a low rate of segregation could lead to problems, and generally the induction of protein expression in a high copy number vector will have deleterious effects on the host, increasing the chances of plasmid loss. Regards, Andrew.
Dear Andrew,
I want to clarify that I have a WT, null and a complement strain where the complemented strain has the kanamycin casette along with my gene of interest. Both my WT and null do not contain any plasmid. There is no burden for protein production as it is from the genome under its native promoter. To generate complemented strain, I have transformed the null cells with a plasmid carrying my gene of interest and to select for the complemented cells I use kanamycin. To maintain homogeneity in experiments, I have transformed both WT and null cells with the empty vector containing kanamycin resistance gene as selection marker. So, now I grow all of them in presence of kanamycin. I hope you have got the question now.
Thanks!