Hello,
I am relatively new to using electroporation transformation. Recently I cam across a problem transforming two plasmids (one with >10 kbp and the other approx. 6 kbp) into MG delta lacI E coli. I had difficulty transforming this plasmids pair using heat shock, and then attempted to use electroporation.
I prepared my electro-competent cells according to an online protocol from Barrick Lab (https://barricklab.org/twiki/bin/view/Lab/ProtocolsElectrocompetentCells).
I used 0.1 cm cuvettes, bio-rad MicroPulser, Ec 1 setting. I got time constant generally above 5.5 ms. But whatsoever no colonies on two-antibiotic plates, even though abundant cells growing well on plates without any antibiotic, suggesting high cell survival after electroporation.
In fact, with electroporation, I couldn't even another control get plasmids pair to be co-transformed (the large plasmid > 10 kbp and a backbone plasmid approx. 3.9 kbp), the pair can be easily transformed with heat shock into MG cells.
I was previously told that time constant above 5.5 ms was pretty good. Since plenty cells survive after electroporation, what could possibly be the reason(s) that made these transformations fail? Thanks a lot!
Did you check that your two plasmids, in addition to having different selection markers, have different replication origins ? Plasmids having the same replication origin are notoriously difficult to maintain in the same strain. Otherwise, if the likelihood of successful simultaneous transformation transformation is equal to the product of the two frequencies for each plasmid, it may explain why the event is very rare: assuming that each transformation event occurs with a frequency of 10exp-5, the frequency of simultaneous tranformation will be (10exp-5)exp2, i.e. 10exp-10. Thus, your best chance of getting a strain harbouring the two plasmids may be to proceed with the two transformations sequentially (assuming that they can be maintained: see above comment about origins of replication)
Hi Pierre, thank you for the reply. The two plasmids I am co-transforming are compatible in origin of replication. One plasmid contains ColEI (pBR322 vector) and the other is pSC101. Sequential transformation indeed seems a safer option to follow.
I have a theory why the co-transformation failed. As I later streaked the survived cells from the blank antibiotic plate into single antibiotic plates, I noticed one plate seemed to show more colonies, suggesting that only one plasmid entered cells (the smaller plasmid) while the other larger plasmid was not picked up. As smaller plasmids are naturally easier to enter, their abundance jammed the cells and prevented cells to pick up larger plasmids. So it is better to adjust the concentration ratio of the two plasmids according to their sizes. I just attempted trial transformations and will know the results tomorrow.
I agree with Pierre Béguin that the simplest way to resolve the problem is to do sequential transformations.
If you can't do that for some reason then I would be sure to do appropriate controls for each of the plasmids singly from your cotransformation experiment. In other words if you are using antibiotics A+B, be sure to also have a plate with only A and one with only B to see if one plasmid is just not transforming well. You should have thousands or tens of thousands on each.
However I disagree with Pierre regarding the frequency calculation (sorry Pierre!), the frequency of co-transformation is much higher than the product of the two single rates. The explanation is that only a small fraction of the cells are "competent", of the competent cells there should be a fairly high rate of co-transformation. I frequently saw 10-20% rates of co-transformation (relative to single transformations) when we looked at it. But if one plasmid is transforming at a very low rate for whatever reason, then the rate of co-transformation will necessarily be less than that single rate.
Quite correct, Michael, I take it back about the probability calculation
I agree that sequential transformation should be the best solution unless the problem lies in the plasmids' load. Maybe there is a gene encoding a toxic product. You could try to transform each of the plasmids individually and compare the transformation efficiency, also be sure both plasmid preps are equally clean and in similar concentration. I double-transformed plasmids of similar size and it worked well; triple-transformation is also possible
Hi Ximing Li,
Your protocol of electroporation transformation is the same as mine.
But, I use small plasmids (one with ~5 kB and the other approx 3 kB) into E. coli DE3 (like E. coli rosetta and E. coli artic express for heterologous expression). They had different selection markers and I have success in transformations.
In my experience, the problem is the size of your plasmid (10 Kb is big).
I agree with the colleges of science. Using a cotransformation experiment is a good idea. And in addition, you can use positive control (one small plasmid ~3 kB).
Best Wishes,
Cássio Siqueira.
Hi, Cássio Cassiano. I have a question regarding transformation of plasmid to e coli that have different antibiotic resistance marker. While transforming plasmid to Rosetta cells (rosetta with pRARE-CamR gene), let's say the plasmid have kanamycin resistance gene, are you inoculating them in the plate that have 2 antibiotic (Cam 34 ug/ml and Kan 30 ug/ml)?
Hi Jonathan Jonathan
Yes. You must use antibiotics in your experiments if the cell has two plasmids. Use Cam and Kan in the correct concentration.
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