In Figure 2, plasmids isolated from E. coli colonies produced during mutation cycles using XL1-Red competent cells were transferred into a non-mutator E. coli host, then plated and incubated on LB/Amp agar plates. From these master plates, colonies were replica plated onto minimal-media agar containing rhamnose (to induce esterase production), substrates 1 or 2, and indicators (crystal violet and neutral red). Upon hydrolysis of substrates 1 or 2, the pH in the microenvironment of the colonies decreased and red spots are formed (from the indicator).
We achieved a good compromise between fast bacterial growth and reliable detection of positive variants by using plates with minimal media. From approximately 750 colonies, several putative mutant clones were identified based on the red color that developed on the agar plates after the colonies were incubated for 2 to 6 days at 37C.
The plasmids were isolated using colonies from the master plates and then transformed back into a non-mutator host. After the colonies were cultivated for 3 hours and induced with rhamnose for 4 hours, the esterase produced was isolated by sonification and subjected to preparative biotransformation. When the esterases capable of hydrolysing the 3-hydroxy ester 1 were sequenced, one variant contained two point mutations (A209D and L181V). In the indicator assay, the clone that produced this variant also gave the strongest red color and largest colony size.
The esterase gene is 843 bp long, and the double mutant was isolated after
three mutation cycles. Each cycle is approximately 30 generations and the
spontaneous mutation rate should yield approximately 1 mutation per 2,000 base
pairs every 30 generations. This PFE mutant was subjected to another mutation
cycle using XL1-Red competent cells, and approximately 9500