Let's say I have one plasmid (pPICZalpha) with one GOI linearized by one RE and I get 20 colonies. Does it make sense to screen them all for expression or is it basically nonsense?
The point is, what can be the expected range of expressed proteins from single transformation? If the average activity would be 100 pkat/ml of media (secreted protein), what range to expect? 80-120 pkat/ml? In that case I really wouldn't care. However, if it would be from say 0 - 500 pkat/ml, in that case it would be already more interesting, not to say if the range was from 0 to several thousands of pkat/ml.
However, the question is, since the integration occurs via homology recombination into one specific place, there is no positional effect (like in plant transformation), so why should one screen more colonies?
(and the manual from Life Technologies does recommend that)
Dear Tomas,
It depends on what strategy you used for recombination. If it is integration strategy by digesting within homologous region (Generally AOX promoter region), YES you must screen more clones (~100-500) to get high yielding clones and you will find at least 7-10 folds enrichment in target expression.
If you used cassette replacement strategy by digesting your construct outside of the promoter region, theoretically you will find one copy of gene/cell and may not reflect yield difference while screening multiple clones.
it was the digestion in the AOX promoter. Unfortunately I don't have that many colonies so I'll have to go with all I have by now.
But I don't understand why should there be any difference if the transformation is via homology recombination?
Dear Tomas,
Check the attached free hand diagram :) to understand the multiple events of gene integration into the pichia genome
Yes.. target expression is proportional to no of cassette (Gene copy number) integrated into the genome.
When we are talking about cassette, it includes [Promoter + Gene of interest + TT + Zeocin]. So try to pick the clones from High zeocin containing plates (1500, 2000 & 2500µg/ml) to get high yielding clones.
So I measured activity in the medium after two days of expression (what I had from previous weekend) and besides three clones which had activity at the level of background (which I do not really understand as they were all confirmed to be positive), the activity ranged from 45 - 80% conversion so I guess no wide range. But I will continue with the seven clones with highest activity and confirm it in bigger volume (now I had only 2ml).
- Badges
- Science topic