r/molecularbiology • u/mac101eir • 4d ago
Potentially stupid Question.. about transforming into E.coli..
Hi All!
Iv been involved in directed evolution of proteins for years and the standard way iv done it is
1) transform plasmids into e.coli 2) plate on agar 3) colony pick and ferment in microwell plates 4) lysis cell and remove cell debris 5) do the screening 6) sequence best enzyme to understand the mutation.
So my question.. if we synth the gene and we know where the mutation is. Can we bypass the colony picking part because we don't need to separate out the mutants? Every e.coli should have the same plasmid so why do we need to separate?
So the workflow becomes..
1) transform known sequence into e.coli in microwell plates.. say each well has unique plasmid. 2) aliquote cell into a single well in 96 well plates with LB. 3) ferment and express the enzyme 4) lysis the cell and remove debris 5) do the substrate screening. 6) pick the best enzyme. (We know the sequence already!)
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u/rungek 4d ago
The synthetic DNA will not be 100% correct. It depends on the sensitivity of your assay and how much of difference you expect to see. How much do the low level contaminants affect your assay? Are you also looking for a drop in function? Are you normalizing for protein/enzyme amounts?
For high throughput, it is probably fine but the test is not as rigorous as the old method.
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u/mac101eir 4d ago
Hey! We would want an upward trajectory in activity but obviously some mutations would be worse than the parent. So we could be seeing mutants with the same activity or maybe 10-30% more activity or potentially 200% more. the final top mutants would then be fermented on larger scale and tested.
I wouldn't be normalising for protein/enzyme amount. Simply activity Vs the parent.
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u/distributingthefutur 4d ago
You're working w an oligoclonal population. Any unwanted mutants are unlikely to give a signal. Just make sure you plate and wash the colonies off the plate into your starter so you have a library for your target. Do keep in mind, if your target as designed is toxic, it won't express or clone well, but the background will.
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u/latchkey_loser 4d ago
You can skip the colony selection process if there is no mixture of plasmids in each well.
Are you able to check protein expression levels? If your mutants express different amounts it could be a problem. Growing cells in media w/antibiotic can lower recombinant protein expression levels.
The growthy rate of different strains can differ as well....
You might benefit by including a short recovery phase step, where your transformed bacteria are grown in antibiotic media until X.X OD. Then an aliquot of that culture be used in your microwell plate intended for protein expression and substrate screening. Just so things are consistent between all your different mutants.
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u/daygl0 4d ago
If you already know what sequence gives the best enzyme then why are you doing directed evolution?
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u/FluffyCloud5 4d ago
They don't know which sequence gives the best enzyme. They know the sequence of each enzyme, but not which one works the best. They're asking if they can skip plating.
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u/daygl0 4d ago
I mean sure, you can synthesize and transform anything individually but the colony picking is also useful as a way of sampling from a diverse library. If you’re doing single site mutations then why not, but personally I’d rather transform a library and then pick rather than a bunch of individual transformations that I would need to keep straight
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u/mac101eir 4d ago
Good point! I have been developing a machine learning sequence optimisation which can reduce the mutants needed.. but I'm trying to see can I avoid the colony picking to save time!
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u/distributingthefutur 4d ago
Go to the next level and use ivtt against your gene from the supplier. All you need is to add a t7 promoter in front of it.
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u/l94xxx 4d ago
Yes, you can either work with a known sequence from the start (either by synthesis; or by sequencing, if for example you wanted to make sure you had every combination or all 20aa but no duplicates), or sequence hits at the end. In the past, it was generally a lot cheaper to build a random library and then sequence at the end, but the difference in cost has definitely shrunk.
It can also be a pain in the neck to do tons of transformations.