I'm trying to transform two plasmids. One from pET-Duet1 vector containing only one gene in the MSC1 region and another from pBB541 vector. Both with different antibiotics selections.
I can surely transform the BL21 competent cell when trying one of the vector separately but when I combine them, nothing works.
Would you guys have any suggestions?
Thank you!
Are you transforming sequentially, by transforming BL21 with plasmid#1 then making competent cells of the resulting BL21(plasmid #1) strain, then transforming plasmid #2 into that strain? Or are you putting both plasmids in at once?
Just checking, because only a small subset of the total number of E. coli are going to take up either plasmid, and the odds that two separate plasmids are going to enter the same E. coli cell are much lower than a single plasmid alone.
There is a very low probability of getting both the plasmids into E.coli. Few things to consider are
1. Size of the plamsids.
2. Concentrations of the plasmids if you are trying to transform it together.
3. If you are trying it sequentially, then there is low probability again. Even if you get a double transformed cell initially, there is a chance that the bacteria extrudes the one of plasmid out eventually , leaving you with false positive results.
Thank you for the response Santanu Sasidharan and Alexandra Johnson !
So, I tried putting them together and doing it sequentially. But I didn't get any results. further than this. Nothing grew on the plates with both antibiotics for selection.
However, I plated the set I mixed both plasmids both in LB with both different antibiotics.
I did it to check whether any plasmid got transformed. It turns out that one single colony grew from this mix in the plate with the antibiotic that selected for the plasmid with my protein of interest. Conversely, hundreds grew from this same mix on the plate with another antibiotic selection for my chaperone.
So is this some sort of a competition between the plasmids?
Do you have any idea of a protocol that could work?
Maybe by taking one already transformed, make them competent again and transform with the second Plasmid? Wouldn't the E.coli extrude the first plasmid when I make them competent again?
When you make them competent again a very small number might get rid of the original plasmid, but it's not a huge worry, especially since you can just select using plates with both antibiotics. Sequential plasmid transformation is a very common thing that's been done for decades.
When making them competent sequentially also make sure you're growing them with antibiotics for the first plasmid for the growth process of making them competent and plate for selection with both antibiotics. Also try transforming your competent cells with the first vector with another compatible plasmid as a control to make sure it's just not an issue with the transformability of your competent cell prep.
Queren Alcantara Can you specify the size of the plasmids that you are trying to transfer?
Thank you, guys for all the answers. I made the cells competent again with one of the plasmids so I could transform the second plasmids just fine. It worked out!
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