hi to all,
i was wandering if it is possible to transform bl21 de3 cells with two plasmids.
the aim is to espress 2 proteins at once.
the two plasmids are both iptg inducible, and ampicillin resistent.
should i use untrasformed cells or could i use cells already tranformed with one of the two plasmids and tranform these cells with the other one?
thank you
Agree with Michael. You can't use two plasmids from the same incompatibility group (controlled by which origin of replication is present, see this link for more info: http://blog.addgene.org/plasmid-101-origin-of-replication) and need to choose plasmids that have different antibiotic resistance markers. If you want to co-express two proteins, you'd be better off making a polycistronic message in E. coli (for reference, see this publication http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2582387/).
Generally if people want to transform with two plasmids, they use plasmids with different origins of replication so that they are stable within the cell. This can be done with two plasmids that are IPTG inducible. However you especially need to make sure they have different antibiotic resistances, otherwise how will you determine whether the strain has one plasmid, the other plasmid, or both?
Agree with Michael. You can't use two plasmids from the same incompatibility group (controlled by which origin of replication is present, see this link for more info: http://blog.addgene.org/plasmid-101-origin-of-replication) and need to choose plasmids that have different antibiotic resistance markers. If you want to co-express two proteins, you'd be better off making a polycistronic message in E. coli (for reference, see this publication http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2582387/).
As said previously you should pay attention to antibiotic resistance and origin of replication, if both are ampicillin resistant it won't work.
For the second part of your question , you can either transform sequentially (one after another) which is longer process, or both at the same time. The first technique should work with any plasmid the second one might not work well depending on the plasmids by heat shock. But if you electroporate there should be no problem at all for co transformation. You could also use plasmid like Duet plasmids that enables cloning of two different gene in the same plasmid with two distinct promoters. Though it seems that often one gene is more expressed than the other one. Or you can clone to make a polycistronic DNA as suggested Daniel above. THe risk is also that proteins are not expressed in the same ratio. But having two different plasmid does not guarantee equal expression as well (though it worked fine for me so far).
Hi there,
Polycistronic approach is easier if you can afford to buy synthetic gene construct.
Hi Antonio,
You can not transform two plasmids having same origin of replication and same resistant markers due to competitive environment caused by the limited resources for the same recognition site, secondly as said by others, there would be enormous ambiguity over the presence of two plasmids due to presence of same resistance markers.
... The only problem to be considered is the compatibility of the plasmid and not transcription ... yes, I would do co-transformation
Hi, I have two compatible plasmids with different resistance markers and I'm trying to co-transform in BL21 (DE3) cells with no succes. Any special protocol for that? Any tip?...competence needed or amount of DNA to be transformed? Thanks
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