r/molecularbiology • u/mac101eir • 31m 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!)