I have been trying to knock-in a designed genetic fragment at TRP1 locus taking advantage of 5FAA counter-selection (which selects for the loss of TRP1).
I used PCR product as the insert, electroporation as transformation method and haploid baker's yeast strain BY4739.
The counter-selection worked because I did get colonies that were tryptophan-auxotrophic.
Integration at TRP1 site occurred but with unexpected knock-in fragment. I used primers upstream and downstream TRP1 gene, which are also at the ends of my knock-in PCR fragment, to check the isolated transformants. While the wild-type control gave me expected size for wild-type TRP1+homology regions, the transformants did not give the expected size of the knock-in: no band in some, smear in others. I only tried 10 isolates.
I wonder what could go wrong? Someone said I need to do this in diploid. Is that necessary?
Something definitely happened to the TRP1 site after transformation, what it could be besides the homologous recombination using my insert as template?
Hi there,
Unless the knock in is toxic for the cell I can't see why it couldn't work properly. In this case working in a diploid strain wouldn't change anything.
What protocol do you actually use? Do you spread your transformation mix on rich media first and then replica plate it onto 5FAA+TRP containing plate? In my experience t's always better to let the time to the cell to recombine first and then to apply selection so that the expected event is actually occuring
The other issue might be the PCR a you don't get signal for some clones: is it because there is no priming allowed or is it because the PCR reaction is not occuring for some reason (quality of the template for instance). Do you actually check template quality with an other set of primers supposed to work properly?
Thanks a lot, Dominique, for your insightful comments!
The knock-in is not toxic to the cells! I simply adapted the electroporation protocol of BioRad MIcroPulser manual, which I have used to transform plasmid with high efficiency.
Worth mentioning, I added 1M sorbitol post electroporation for recovery and incubated at 30C for 1 hour before plating onto minimal media plus 5FAA and trytophan. Wouldn't plate directly onto rich media result a lawn which might cause problem for downstream selection? How about adding YPD post electroporation
The PCR product was from fusion PCR and had multiple bands. The strongest band is of the expected size though. I did not gel purify to keep high DNA concentration so that I could add ~500 ng to 50 ul cells. Next I will definitely purify before electroporation.
One of the check primers was from outside of the knock-in insert which means it was never used in PCR reaction for insert construction. So the check PCR should be fine.
I PCR checked 40 isolates newly and found most of them gave wild-type size.
I will redo the experiment using purified PCR and recovering with YPD.
Thanks again!
Hi again,
What I call a toxic knock-in is cell toxicity due to the expression of the gene contained in the inserted cassette. About time for recovery after transformation, it is always advised to do so (at least a few hours in rich media without selection) before plating. In your case a double recombination event is expected : I always go for plating transformation mix on YEPD plate first and replica plate the day after onto selective media (no lawn observed as incubation is just overnight). Furthermore 5FAA selection is better at 0.5g/L in complete minimal media containing 5% glucose (according to this article :http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0061(200004)16:6%3C553::AID-YEA554%3E3.0.CO;2-7/full)
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