My host cell, which is E.coli AS17/pBAD-Rv1495, already has a plasmid inserted that expresses the Rv1495 toxin. I made these cells electrocompetent and proceeded to insert another plasmid of interest (pLIC-MtopWT) using the electroporation method. I successfully managed to acquire a lot of colonies upon plating these cells into their respective antibiotic agar plates, which is suppose to indicate the presence of these two plasmids. I selected a handful of colonies and re-plated them in new plates in order to label them and test their activity. When I saw that a colony indeed showed the expected activity in the first assay, I proceeded by repeating the assay with a new cell culture from the SAME colony. However, it seems that the results for the second assay showed no activity, even though the first assay did. I've been repeating the assays and it seems arbitrary how sometimes the activity seems to be present in some assays and in other assays it is not. I am having trouble finding literature that might give an insight as to what the problem might be when it comes to doing transformation with multiple plasmids. What kind of troubleshooting might I be able to do to figure out what is happening in this case?
Rosemarie Martinez
Why are you double transforming?
I think you need to first understand the properties of the two plasmids because there might be some kind of plasmid incompatibility. This happens if the two plasmids have the same origin of replication as this might prevent them from being passed on together in one cell hence you end up with two homogeneous populations with either plasmids.
Antibiotic selection is not a confirmatory step for you to conclude that double transformation was successful. You need to confirm using one or two of the following methods;
1. Colony PCR using specific primers for inserts of each plasmid
2. Restriction digestion
3. Confirm by sequencing
Thank you and best wishes
Fortunate
It depends if your plasmids have different/compatible replicons. pLIC is pBR322-based. I don't know what the replicon of your pBAD is. What was the empty backbone you used (pBAD33, pBAD24, pBAD/His) ?
Adding on Nathan Fraikin answer, beside compatible origin of replication make sure you have two different selection markers (i.e. two different antibiotics resistance).
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