Dear all,
due to a couple of rare codons in the sequence of my protein of interest, I am using BL21 Codon Plus RP for expression from a pRSET A vector. Because the pRSETA conveys ampicillin resistance, I use ampicillin+chloramphenicol plates (150 µg/ml Amp/35 µg/ml Camp) after transformation, but when I pick these colonies and inoculate them in the medium which contains ampicillin+chloramphenicol (150 µg/ml Amp/35 µg/ml Camp) then the cultures fail to grow (remain clear even after 24 h of incubation)...
Any advice in this issue would be very much appreciated :)
Note: I am using minimal plates and medium (imperative, for isotopic labelling).
Note: Normal BL21(DE3) (ampicillin only) grows faster on minimal plates (12 hours on 150 µg/ml ampicillin-only compared to well over 24 h for the CodonPlus-RP on amp/camp double-selection). So maybe the combined load of antibiotics is too high in the latter case? Is this a common observation with CodonPlus?
Best regards,
Peter
Maybe you try to grow the transformants in minimal medium containing 100 and 25 ug/mL Ampicillin and Chloramphenicol respectively.
Dear Peter,
I think that the absence of growth is coming from the too high concentrations of antibiotics; for ampicillin, 100 µg/mL is a maximum for E. coli and 50 is even sufficient to maintain the plasmid; for chloramphenicol, 30 is generally recommended. Did you check the colonies on plates (I mean by PCR on colonies) ? Definitely, for me, you must lower the concentration in antibiotics, good luck
Colette
Hi Peter, well theoretically you shouldn't experience what you experience... unless the pRSET A plasmid, which is a relatively low copy plasmid, is not allowing you to use 150 ug/ml Ampicillin during growth, so the suggestion to reduce to 100 ug/ml could be good, then you can raise it again during expression to stop growth, force keeping the plasmid and maximize expression. For the Chloramphenicol you should be fine, you can go up to 50, but 30-35 ug/ml are no problem at all.
Just as a comment, colonies always grow better, because they are protected from the antibiotic by the first bacterial layer on the agar, but in solution all cells are exposed to the same concentration of the antibiotic, so the conditions are more stringent.
Thanks Pietro, that was exactly what I thought, it should have worked out, since other expressions with the same load of antibiotics worked out fine (but these were normal BL21(DE3)) - I now suspect my construct may be toxic...
I am now doing a big experiment, testing my construct and a control plasmid on multiple strains with a lot of controls... Should enlighten the case.
If anyone bothers: It turned out that my expression plasmid is toxic when used in the CodonPlus strains (No growth in RP or RIPL, but normal to excellent growth in BL21(DE3) and the pLysS derivative).
Antibiotics were perfectly tolerated at 150 µg/ml (Ampicillin) and 35 µg/ml (Chloramphenicol).
Interesting! I wonder if the protein is/was toxic towards Codon Plus strains, why Codon Plus transformants (with plasmid expressing gene for this toxic protein) got selected on LB agar plates in the first place?
Peter, you can isolate pRP/pRIPL plasmids from the Codon Plus strains and transform into BL21(DE3)pLysS. If the product of the gene was indeed toxic in presence of rare tRNAs, the BL21(DE3)pLysS strains expressing both your gene of interest and rare tRNA should not grow.
A couple of rare codons should not make such a drastic difference. Does this mean that protein being expressed in BL21(DE3)pLysS is non-functional (hence not toxic)?
Could it be that expression of your protein somehow renders bacteria more sensitive towards chloramphenicol.
Indeed interesting, but not so much that I would investigate more ;)
Chloramphenicol sensitivity cannot be the explanation, since pLysS also conveys Chloramphenicol-resistance...
Something about the CodonPlus, making them weak somehow and causing slow growth. BL21(DE3) unaltered once again proves to be most robust.
I am going to mutate the codons I am bothering about and that should be it. :)
The BL21(DE3)pLysS were maintained in presence of Chloramphenicol?
I have worked with rare tRNA expressing vectors for most proteins I work with contain multiple rare codons even in tandem but have not observed significantly slow growth in presence of rare tRNA.
Yip, BL21(DE3)pLysS grew in Amp/Camp at the concentrations stated above to ODs comparable to BL21(DE3) in just Amp.
Other constructs work nicely with RP or RIPL in my hands as well - Just this particular expression plasmid only seems to like "normal" BL21(DE3).
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