Nitrilases, nucleic acids encoding them and methods for making and using Them
A technology of nitrilase and nucleic acid, applied in the fields of biochemistry, chemistry, and molecular biology, can solve the problems of not being able to participate in high-throughput screening, troubles, etc.
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Embodiment 1
[0340] Example 1: Phagemid Infection
[0341] For each library that was used to screen nitrilase, infections were prepared as follows: 5 ml of OD of SEL700 cells 600 The nm=1 resuspension was mixed with 1 ml of the phagemid library to be screened. The composition was incubated in a water bath at 37°C for 45 minutes.
[0342] Using the infection, in 10mM MgSO 4 Serial dilutions were performed in , using 10 μl aliquots of the infection.
[0343]
[0344] Place 60 μl of each of the following dilutions into a small LB-Kan 50 On tablet:
[0345]
[0346] Centrifuge the cells in the infection in a benchtop centrifuge at 4°C, 4.6k rpm for 10 min to form a pellet. The supernatant was decanted from the resulting pellet. Resuspend cells in remaining liquid. Place all resuspended cells into a single large LB-Kan 50 on the tablet. All plates were incubated overnight at 30°C.
Embodiment 2
[0347] Example 2: Selective Screening
[0348] With ∼4 ml 10mM MgSO 4 Resuspend the cells on each infection plate. Place the resuspension into a test tube. With ~3 mL of 10mM MgSO 4 The remaining cells on each plate were resuspended and combined with the first resuspension from the same plate. with 10mM MgSO 4 The volume of each tube was brought to 12 ml and the tubes were vortexed vigorously. Centrifuge the tubes in a benchtop centrifuge at 4°C, 4.6k for 10 min to form a pellet. From each resuspension, decant the supernatant. with 10ml 10mM MgSO 4 Resuspend the washed cells in each tube. Resuspensions of each library were stored at 4°C until ready to establish selective cultures.
[0349] For each resuspension, establish a selective culture using the following procedure:
[0350] 1) Prepare nitrilase selective medium using 1XM9 culture including 0.2% glucose, no nitrogen, and 50 μg / ml kanamycin (only for pBK phagemid library; for pBS library, use ampicillin) base...
Embodiment 3
[0356] Example 3: Isolation of positive nitrilase clones from selective cultures
[0357] Streak ten (10) μl of the growing selective culture into a small LB-Kan 50 plates and allowed to grow for two nights at 30 °C. Five isolated colony forming units (cfu) were picked and each of them was grown in 2 ml of nitrilase selective medium at 30°C. Each culture was monitored (where growth indicated positive colony forming units were picked) and removed when monitoring indicated it was in a stationary phase of growth. One (1) ml of culture was used to prepare phagemid preparations and eluted with 40 [mu]l of elution buffer. Five to eight (5-80) μl of DNA are spliced with Pst I / Xho I or Sac I / Kpn I restriction enzymes to remove the insert from the vector. A restriction fragment length polymorphism (RFLP) assay is done to identify the size of the insert. Inserts are sequenced.
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