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Nitrilases, nucleic acids encoding them and methods for making and using them

A nitrilase, nucleic acid technology, applied in the fields of molecular biology, biochemistry and chemistry, which can solve problems such as troublesome and inability to participate in high-throughput screening well

Inactive Publication Date: 2006-10-18
VERENIUM CORP (US)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, it is a cumbersome method that does not lend itself well to high-throughput screening

Method used

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  • Nitrilases, nucleic acids encoding them and methods for making and using them
  • Nitrilases, nucleic acids encoding them and methods for making and using them
  • Nitrilases, nucleic acids encoding them and methods for making and using them

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0337] Example 1: Phagemid Infection

[0338] [000268] For each library that was screened for nitrilase, the infections were prepared as follows:

[0339] OD of 5ml 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.

[0340] [000269] Using the infectious agent, in 10mM MgSO 4 Serial dilutions were performed in , using 10 μl aliquots of the infection.

[0341] Library Titer Perform Dilution

[0342] ~10 5 cfu / ml 10 -1 dilution

[0343] ~10 6 cfu / ml 10 -1 , 10 -2 dilution

[0344] ~10 7 cfu / ml 10 -1 , 10 -2 , 10 -3 dilution

[0345] [000270] Place 60 μl of each of the following dilutions into a small LB-Kan 50 On tablet:

[0346] Library Titer Perform Dilution

[0347] ~10 5 cfu / ml undiluted infection, 10 -1 dilution

[0348] ~10 6 cfu / ml 10 -1 , 10 -2 dilution

[0349] ~10 7 cfu / ml 10 -2 , 10 -3 dilution

[0350] [000271] Centrifuge t...

Embodiment 2

[0351] Example 2: Selective Screening

[0352] [000272] With ~4ml 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.

[0353] [000273] For each resuspension, a selective culture was established using the following procedure:

[0354] 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, ...

Embodiment 3

[0360] Example 3: Isolation of positive nitrilase clones from selective cultures

[0361] [000274] Streak ten (10) μl of 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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Abstract

The invention relates to nitrilases and to nucleic acids encoding the nitrilases. In addition methods of designing new nitrilases and method of use thereof are also provided. The nitrilases have increased activity and stability at increased pH and temperature.

Description

[0001] Cross References to Related Applications [0002] [0001] This application claims the benefit of priority based on U.S. Patent Application Serial No. (USSN) 10 / 241,742, filed September 9, 2002, and USSN 10 / 146,772, filed May 15, 2002, both of which applications claiming priority benefits to USSN 60 / 351,336 filed on January 22, 2002, USSN 60 / 309,006 filed on July 30, 2001, and USSN 60 / 300,189 filed on June 21, 2001; And, this application is a continuation of USSN 09 / 751,299, filed December 28, 2000, which claims USSN 60 / 254,414, filed December 7, 2000, and USSN 60, filed December 29, 1999 / 173,609 of the priority benefits of each of the applications. These applications are hereby incorporated by reference in their entirety for all purposes by reference to the subject application. [0003] Copyright Notice [0004] [0002] Pursuant to 37 C.F.R. §1.71(e), a portion of this patent document includes material that is subject to copyright protection. The copyright owner has no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00A61K38/44C12N1/20C12N1/21C12N9/78C12N15/00C12P7/42C12P13/00C12P13/04C12P21/04C12P21/06C12P41/00C12Q1/00C12Q1/34C12Q1/68
CPCC12N9/78C12P7/42C12P13/004C12P13/04C12P41/006
Inventor G·德桑蒂斯J·M·肖特M·伯克K·王R·法韦尔K·查特曼
Owner VERENIUM CORP (US)
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