Potassium Chloride → L-tert-Leucine

A gene encoding the leucine dehydrogenase from B. stearothermophilus is subjected to mutagenesis by error-prone PCR according to the method of May et al. The error-prone PCR is performed in a 100 mL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer (10 mM TRIS, 1.5 mM MgCl2, 50 mM KCl, pH 8.3), and also containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase. Conditions for carrying out the PCR are as follows: 2 minutes at 94° C.; 30 cycles of 30 sec 94° C., 30 sec 55° C.; 2 minutes at 72° C. The PCR product is double digested with Nco I and Bgl II and subcloned into pBAD/HisA vector which has been digested with the same restriction enzymes. The resulting leucine dehydrogenase mutant library is transformed into an E. coli host strain LMG194 and plated on LB agar supplied with 100 μg/mL ampicillin. Individual transformants are inoculated into 96-well master plates containing 0.2 mL LB Broth with 100 μg/mL ampicillin, and growth is allowed to take place for 8–16 hours at 37° C. with shaking at 200 rpm. Each well in each master plate is then re-inoculated by a replica plating technique into a new second stage 96-well plate pre-loaded with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5–10 hours at 37° C. with shaking at 200 rpm. The second stage plates are then centrifuged at 14,000 rpm for 20 minutes, and the supernatant is decanted. The cell pellet in each well is washed with 200 mL of water. The washed cell pellet is suspended in 30 mL of B-Per. After mixing, the suspension of cells in B-Per reagent is allowed to stand for 10 minutes at room temperature, and a solution having the following composition is then added to each well in the plate:

1. customis performed in a 100 mL reaction mixture
2. additionalso containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase
3. custom2 minutes
4. customat 94° C.
5. custom30 cycles of 30 sec 94° C., 30 sec 55° C.
6. custom2 minutes
7. customat 72° C
8. customfor 8–16 hours
9. customat 37° C.
10. waitto continue for 5–10 hours at 37° C.
11. stirringwith shaking at 200 rpm
12. customthe supernatant is decanted
13. washThe cell pellet in each well is washed with 200 mL of water
14. additionAfter mixing
15. additionthe suspension of cells in B-Per reagent
16. waitto stand for 10 minutes at room temperature
17. additionis then added to each well in the plate

ORD record

ord-6278e0ff00454b8db263fd1d9c2b0e04

Source dataset

uspto-grants-2007_04

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US07202070B2