Dear all,
Having done many overnight cultures of various strains of E. coli with various vectors I was surprised to come across so much trouble in my current project.
My project uses the yeast-expression vector pPICZalpha (A,B,C) to express genes in Picha. However, the TOP10 E. coli propagating this vector (empty OR containing a gene of interest) takes 2 days (instead of 16 h) to reach a good enough concentration for mini/midi prep. Anyone else have the same issue?
I follow the protocol to the T: low salt LB, appropriate Zeocin concentration. 20 mL falcon with 5 mL of LB, shaking at 37C. On one occasion I checked the OD before miniprep, it had a good value, but the miniprep yield was poor. As far as I can tell, pPICZalpha is not a low-copy number plasmid.
My solution is simply to grow the bacteria for 2 days, so it's not a disaster, just a waste of time. If anyone had a similar problem and managed to solve it, let me know!
All the best,
Emma
I've not used the vector, but could it be expressing something else that is toxic to Top10 cells? These cells normally grow like mad!
Alternative is to try removing zeocin and see if your cultures grow faster? Though you won't get selection for the vector... but worth a try to see if it is zeocin that is stunting the growth.
Dear Emma,
Why don't you try to use 2YT (16g/L Tryptone, 10g/L yeast extract, 5g/L NaCl) and 25 ug/L zeocin? In my hands this works well.
Hi Emma, I read through your problem, I having the same issue here. Did you find the solution? Did the suggestions from above work? Thank you in advance.
@Bernardo Hi, I'm sorry you're having this problem too! Unfortunately I never found a good solution. My advice would be to grow large cultures of your E. coli to get a sensible amount of product.
I have since left this Pichia project, but if you crack this case, please share!
Best, Emma
OK! Thank you for the advice Emma. Yes, if I find a better way to do it I will report it.
Best, Bernardo
The amount of zeocin seems to be the limiting factor here. Although it's suggested to use 25 ug/ml of Zeocin for prokaryotes you should always remember to store the plates and media containing zeocin in the dark because of it's light sensitive nature.
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