I am trying to make a clone using E coli as host and use that clone later to transform budding yeast. But, I am not able to get clone somehow so I was wondering to perform lithium acetate transformation in yeast to assemble my insert and vector directly in it. How successful is a direct cloning in yeast?
I've no direct experience of cloning in yeast so cannot give an answer to the main question here. However, if you want to discuss the cloning problem you're having in E.coli I'm happy to try to help.
Hey Robin, I appreciate your response.
I am trying to insert a polymerase (mono ADP-ribose polymerase) into a pGEV vector under gal promoter using gibson assembly. When I transform E.coli with this construct (assuming a successful gibson assembly), I plate them on LB Carb + "1%Dex" to repress gal promoter. The colonies are constantly negative for the desired construct. Can you help in listing out the possible variations in this cloning that I can try? Let me know if you need any clarification.
You're welcome.. I must confess I am not a big fan of Gibson (being a traditionalist) ; it has some big strengths for certain applications but in my day job I undertake a lot of corrective cloning using standard restriction / ligation methods to fix projects where people have used similar "new cloning" methods that have simply not worked.
Do you have any scope to use a more conventional method as a backup to your primary cloning plan, or is pGEV not amenable to this? If there is any sequence info that would be useful.
Since I have constructed the mutant form of this polymerase into the same vector and transformed using the same conditions, I got positive colonies, so I expect that Gibson might not be the issue. It could be that functional polymerase due to some leaky expression might be killing the cells. what do you think?
Leaky expression is certainly a possibility, especially given the other evidence. I wasn't thinking earlier when I was reading the details - I'm guessing 1% Dex is dextrose, i.e. Glucose that is used to suppress leaky expression from lac promoters. When you perform the transformation of the E.coli, normally after heat-shock we add LB and then incubate at 37 for a while to allow the cells to recover before plating. Did the LB you used here also contain glucose? Couple of possibilities I can think of - try 1% dex/glucose in the recovery LB as well as the plate ; increase the %Dex to 2% on the plates ; grow the plates at a lower temperature for longer?
I used to transform yeast a lot, but we never used it directly for cloning. When we had issues with a potentially "toxic" construct, we initially tried out a different E. coli strain, before changing our cloning strategy.
Direct cloning in yeasts works, just use a lot of vector (500ng for example) and PCR amplified insert made in 100ul reaction. Make sure that insert have 50bp overlaps withthe vector. Longer overlaps would be better, but you would have to order purified oligonucleotides. Make sure that cells for transformation are fresh from the mid-log culture.
Robin Dickinson
I do add 1% Dextrose while recovery and in plates. I agree with the idea of increasing to 2% Dextrose and growing at a lower temperature. Is 30 degrees Celsius low enough?
Patrizia Stohn
Hey Patrizia, thank you for your response.
Currently, I am using E.coli DH5α chemical competent cells. Can you suggest other strains that might have worked for you?
Pawel Bieganowski
Thank you for providing detailed information and suggestion. Since, the transformation mix I prepare has capacity of 50ul volume for foreign DNA in total of 350ul, adding 100ul instead of 50ul will not make much difference in activity of components in transformation mix. What do you think?
You can precipitate your PCR product to decrease volume if this is a problem. About adding glucose to the medium, thiw would make sense if you clone into E.coli expression vector, most of them have lac repressor binding site. This is not the case with a yeast expression vectors, unless you have the special one that has sequence for the bacterial repressor inserted between yeast promoter and start codon of your cloned gene.
Pawel Bieganowski
The vector that I am using is a replicating one. Will it still make difference?
You have to use a replicating vector to make cloning. Both 2-micron or CEN-ARS should work equally well for cloning. if you want to rescue recombined plasmid by transformation to bacteria (if you want to sequence it) 2-micron is better because it is high copy. For some obscure reasons rescue procedure from yeasts is rather inefficient. It is easier if you have more copies / cell of the plasmid.
Jyoti Sharma
I have worked with DH5-alpha competent cells as well. The other strains used in my former lab were XL1-Blue, BL21(DE3) and JM109.
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