I have to make a screen, based on 1 hybrid assay in bacterial cells. Technically, I just need to co-transform 2 plasmids, the substrate, and the binding protein plasmid and select for the phenotype on the plate with 2 antibiotics.
I worry that I can lose a lot of transformants because they will either the substrate plasmid or the binding protein carrying plasmid. My substrate library contains a high variability of sequences, and to cover it X100 at least, I need to make the co-transformation as efficient as possible.
There is a way to make competent cells with the binding protein plasmid already because it al the same in one transformation reaction, but on the other hand, I have 77 binding proteins to screen, meaning 77 batches of electrocompetent cells. It would be a lot of work, and I would like to know, does it worth it, or not?
So, what would be your advice for the highest efficiency of co-transformation: 1) competent cells with 1 plasmid of interest or 2) to use some tips and co-transform to plasmids with different dilutions?
The frequency with which markers separate, that is, (1 − CTF), is a measure of distance between markers. For example, if two markers show 20% linkage, they might each transform 1% of the cells in a population, but 20% of the cells transformed for one marker (0.2% of total cells) will also carry the other marker. If the markers are not closely linked, they will be separated by the dispersive processes and enter cells separately and randomly. For an individual marker transformation frequency of 1%, the CTF would be 0.01% of total cells (or 1% of cells transformed for one marker would be transformed for both markers). CTF refers to co-transformation frequency. It is a useful index of transformation efficiency.
Hi Yulia Ermakova,
It depends on how large the library size is. (What is the theoretical size?)
Like you said, I would normally prepare electrocompetent cells (EC) already containing one of the required plasmids whenever I need to use multiple plasmids for screening/selection, but that will be a lot of efforts for 77 EC. (I have done this for some 10 variants or thereabout in the past though, when the efficiency was of high importance).
Having said that, you can maximise the efficiency of the EC for co-transformation by observing some tips. An important tip is to make sure that the prepared EC is very dense, with an OD600 of at least 1.8 to 2.0 for a 1:100 dilution. That could potentially also mean that you would have to use more plates for the screening after transformation.
You can check out the two protocols below, perhaps you would find them useful. And you can easily harmonize the two protocols...
http://cshprotocols.cshlp.org/content/2020/6/pdb.prot101220.full.pdf
(In case you do not have access to this, an older version of the protocol can be found at http://cshprotocols.cshlp.org/content/2006/1/pdb.prot3933.full)
http://www.ispybio.com/search/protocols/electroporation_3.pdf
Maybe you can also check if commercial EC can satisfy your need considering that you have many proteins to screen.
I once co-transformed three standard sized (6-10kb) plasmids into E.coli and only selected for one (i wanted to get rid of the two other plasmids).
Surprisingly, out of 40 checked colonies, 38 carried all of the three co-transformed plasmids. So it was quiet efficient.
However, i did it only once and the efficiency might change with plasmid size and antibiotic selection type.
Thank you, Abeeb Abiodun Yekeen ! It was nice to return to the initial protocols and recall some parameters. My pain is the size of the substrate library - it has 10^6 individual variants. And it means that I need to have 10^(6)*100 = 10^(8) transformants. So even I do my competent cells close to the quality of commercial ones (this I can), I need to transform 1 ug of the substrate plasmid. I've never had problems making EC of XL1Blue, XL10Gold, BL21DE3, DHa and etc, but the cells I have to use in this experiment are my homemade strains with 8 genomic mutations, and they are not so perfect as classic strains.
Yulia Ermakova Then for a library of that size, and the way you've described the nature of the strains you are working with, I think co-transformation might not be a good idea. Perhaps it's better to go through the stress of making EC that already contain the substrate plasmid and ensure an efficient screening process, as this might be vital for the identification of desired mutants anyways.
However, I'm wondering if there's a possibility of using some criteria to narrow down the 77 proteins to a rather more manageable number, or at least to create a preference list and prioritize the lead ones. You may then revisit the others later after working with the top ones (if this approach is of any help though).
Also, do you mean that you intend to transform up to 1 ug of plasmid? Isn't that too much an amount for transformation, especially for electroporation?
Abeeb Abiodun Yekeen , maybe I would do little aliquots of EC simultaneously, making it feasible. I have control proteins to check the method; there are only 4 proteins with substrates published. I also can try to compare technics, but I am afraid to end up with 77 EC batches... And in the worst case, I made a substrate library 2 orders less:) In my experience, I made libraries of 1.6^(7), but it was a classical strain and only one plasmid.
1 ug would not be one transformation; it would be around 20 individual reactions of 50-60 ng/cuvette. It can be 10 ng because the efficiency at this amount of DNA is higher, but from 100 ng in a single reaction, I get more colonies because frequency compensates efficiency up to 100 ng. Thank you for your advice. In my department, there are only epigenetic and neurobiology guys, and there is no chance to get a piece of good technical advice!
David Scherf Thank you, David! It sounds encouraging if I would be able to convenience my strains to do the same! :)
Thank you Maurice Ekpenyong ! It's interesting to follow your idea of estimation of the transformation efficiency. But I need more details to make sure that I got it right. So, if I have 2 plasmids with different antibiotic resistance, different origins of replication, and very distinct copy number (1 has up to 700 copies per cell and another one is maintained as a 1-2 copies of plasmid per cell), how should I learn, what is my CTF? Or it will be just a proportion, how I mix these molecules, like 1:10, one to 1:500?
Like David Scherf, I have successfully transformed three plasmids at the same time. I think that the success probability of transforming two compatible plasmids simultaneously should be higher. Of course, the size and resistance of the plasmids have a certain impact on the transformation efficiency. You can try to figure out the ratio of plasmid concentration and using electrocompetent cells will increase the efficiency.
Thank you Roger Davis ! I also was worried, that if I make competent cells with 1 plasmid, I have to grow the culture of competent cells with antibiotic, and it decreases the future transformation efficiency (I was making competent cell for iRFP, and it needed heme oxygenase pre-expressed in the cells, and it was the case). So maybe the good ratio of plasmid in the mix would make my life easier. Thank you for sharing your thoughts!
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