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The resulting colonies were picked and grown in 96-well deep well plates.> -wrap-foot> NotNot detectable Microscale cultivation and expression in 96-well deep well plates was done according to Weis et al. (2004) with some modifications.

This was done in duplicate to create two sets of six 384-well deep well inoculated diamond plates.

Because of this relatively high and uniform expression level the, 96-well deep well cultivations can be used to screen for mutated enzymes produced by semirational design approaches.

Expression levels of both constructs with the native signal sequence in 96-well deep well cultivations were similar and in the range of 0.3 U mL−1.

All resulting constructs were transformed into P. pastoris strain CBS 7435, cultivated in 96-well deep well plates, and screening for PDH activity (Table  2).

Single colonies from these plates were arrayed into 96-well deep well plates containing 200 μL of LB and 100 μg/mL ampicillin and grown for 18 h at 37 °C while shaking at 250 rpm.

The standard deviation within one group of constructs was always approximately 10%, which shows the uniformity of the 96-well deep well plate screening and proves its suitability for high-throughput screening.

5 μl of each culture was used to inoculate 1 ml of 2 × TY containing 100 μg/ml ampicillin and either 0 μM or 36 μM tetracycline in 96-well deep well blocks.

120 μl of lysate were transferred from pelleted culture blocks into 240 μl 384-well deep well diamond plates containing 90 μl per well 100% isopropanol using a 384-well Hydra pipetting instrument (Robbins Scientific).

After the sixth round of selection, clones from rounds 3 6 were grown overnight in 96-well deep well plates with 2xYT broth supplemented with carbenicillin and M13K07 helper phage (10 iu/mL).

Single colonies were inoculated into 800 μl of LB media containing 50 μg/ml ampicillin in 96-well deep well plates, covered with sterile, breathable film and incubated at 37°C on a shaking platform overnight.