Some context:
I'm trying to generate a clone possessing a gram positive phage endolysin gene in E. coli DH5alpha.
I'm using the pet28 vector and initially tried to clone/transform directly into both JM109(DE3) and pLysS with no success.
I can confirm the ligations are working with amplification of the cloning site.
I have even tried fusing the gene to GFP, constructing the pet28_GFP vector first and once I try adding this endolysin I fail to get clones.
Can a gene lead to complete plasmid instability in cloning strains that doesn't possess T7 RNAP which T7 vector systems rely on for expression? I am aware of T7 lysozyme repressing T7 RNAP and maybe there is a possible of this gene performing a similar role
I'm aware the protein is toxic but surely one should be able to generate a library to see it all go wrong in expression attempts.
Could my problems lie in DNA secondary structures effecting plasmid stability (replication)? any insights would be great.
I would suggest you first try with a tightly regulated system like a plasmid using the arabinose promoter (pBAD). These are as close to "off" as you are likely to get with a plasmid system.
Secondly, does your endolysin have a signal sequence on it? If so, then it is likely to be highly lethal. If not, then I would think E. coli can tolerate it.
There are lots of reasons you don't get a cloning to work from PCR issues, to ligation issues to poor vector quality to poor transformation. Have you controlled all of those steps?
I think it most likely the problem is either technical issue or lethality, I would not worry about secondary structure or T7 mimicry, those are highly unlikely. Lastly the pET plasmids are not dependent on the T7 system except for expression, the plasmids replicate fine in virtually any E. coli strain.
Thanks Michael J. Benedik for your insight and suggestion, it does have a signal sequence and by your suggestion highly toxic. Is this toxicity likely disrupting cell envelope synthesis? I am giving the rhaBAD plasmid system a go will post the outcome on this thread.
To mention I have controlled for all the steps in cloning, evening prolonging recovery after transformation and lower incubation temps.
I think the issue is that if you have a signal sequence on the protein then it is going to be secreted to the E. coli periplasm where it will have access to the peptidoglycan resulting in lysis. But if the protein was retained in the cytoplasm then there is no target for the endolysin to act on.
If you use the example of phage lambda, expressing the R endolysin is not toxic to the cell except if co-expressed with the holin S protein that disrupts the membrane and releases the endolysin to the periplasm.
So perhaps if you expressed your protein without the signal sequence then you could get stable protein in the cytoplasm that is not toxic.
But it may also work well enough using a more tightly regulated system like araBAD. It is my experience that pET plasmids are not tightly regulated although pLysS does help. Adding glucose during growth might also help.
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