Reacción #2075708

Ecuación de reacción

leucine

ampicillin

leucine

ampicillin

arabinose

→

Acetophenone

Condiciones de reacción

Tratamiento posterior

1. 1
   Otrois performed in a 100 CL reaction mixture
2. 2
   workup.DISSOLUTIONas template dissolved in PCR
3. 3
   workup.ADDITIONbuffer containing 0.2 mM of each dNTP, 50 pmol of each primer and 2.5 units of Taq polymerase
4. 4
   Otro2 minutes
5. 5
   Otro30 cycles of 30 sec 94 CC, 30 sec 55 CC
6. 6
   Otro2 minutes
7. 7
   Otrofor 8-16 hours
8. 8
   workup.STIRRINGwith shaking at 200 rpm
9. 9
   workup.WAITThe second stage plates are then centrifuged at 14,000 rpm for 20 minutes
10. 10
    Otrothe supernatant is decanted
11. 11
    LavadoThe cell pellet in each well is washed with 200 CL of water
12. 12
    workup.ADDITIONAfter mixing
13. 13
    workup.ADDITIONthe suspension of cells in B-Per reagent
14. 14
    workup.WAITto stand for 10 minutes at room temperature
15. 15
    workup.ADDITIONis then added to each well in the plate

Procedimiento

A gene encoding 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 CL reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PCR buffer 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 CC; 30 cycles of 30 sec 94 CC, 30 sec 55 CC; 2 minutes at 72 CC. 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 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 CC 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 CC 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 CL of water. The washed cell pellet is suspended in 30 CL 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:

Fuente