We recently obtained the BLR(DE3) strains for some experiments. AFAIK is identical to BL21(DE3) but recombination-deficient. However, we appear to be unable to transform the plasmid pJET with our insert into it. We can transform other plasmids like pETDuet with inserts, but not pJET.
We can transform pJET into strains BL21(DE3) and TOP10, but not into BLR(DE3).
Anyone know why these appear to be incompatible? We are puzzled.
Mark- maybe these cells have increased sensitivity to low levels of the eco47IR toxic gene in the pJET vector. Not all inserts interrupt the reading frame. Are you sure this is the case for your insert? Are your BLR(DE3) transforming successfully with a control plasmid?
Mark- maybe these cells have increased sensitivity to low levels of the eco47IR toxic gene in the pJET vector. Not all inserts interrupt the reading frame. Are you sure this is the case for your insert? Are your BLR(DE3) transforming successfully with a control plasmid?
Hi Daniel,
thanks for the suggestion - however, the insert is over 1.5 kb and has stop codons in all three frames; so I think this can not be the explanation...
Yes, we can transform all other plasmids we tried into the same strain, and we can transform the plasmid in all other strains we tried. Both chemically made competent BLR(DE3) cells and electro-competent BLR(DE3) cells were tried.
Mark
Mark- There is at least one report in the literature of problems with plasmid stability in BLR cells in the context of IPTG-induction (Appl Microbiol Biotechnol. 2007 Sep;76(4):795-800. Identification of Escherichia coli host cell for high plasmid stability and improved production of antihuman ovarian carcinoma x antihuman CD3 single-chain bispecific antibody.). However, it seems you are observing a problem at the plating stage, prior to induction.
Do any of the pJET plasmids work in the BLR cells? If none do (i.e. the nature of the insert doesn't matter), then I'd be worried that something strange is going on related to plasmid incompatibility. Could the competent cells somehow already have picked up another plasmid harboring the pMB1 origin of replication? Might be worth screening them by PCR to check for evidence of such contamination.
Beyond that, I'm as stumped as you! Be interested to hear if others have had this experience, as well.
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