反应详情

One set of 800 colonies (Example 2), representing a Genomic Library of Agrobacterium 80/44-2A DNA in the plasmid pCP19 in E. coli AG1, was screened for the ability to convert N-carbamoyl-p-hydroxyphenylglycine (N-carb.) to D(-)p-hydroxyphenylglycine (D(-)HPG). Cells from half of one plate (i.e. from 25 different clones) of the library were scraped off into a microcentrifuge tube containing 800 μl of assay buffer. The assay buffer was a solution of 1% N-carb., 10 mM phosphate buffer, 0.0012% phenol red (0.6 ml of a 2% solution per 100 ml of N-carb. solution) adjusted to pH 6.7 with NaOH. This solution was a yellowish colour. The contents of the tube were mixed, and the tube was incubated in a water bath at 42° C. for 24 hours. Conversion of N-carb. to D(-)HPG results in production of ammonia (FIG. 2) which raises the pH and can be detected by a change in the colour of the phenol red from yellow to pinkish-red. One of the 32 tubes changed colour to pinkish-red. The 25 colonies corresponding to this tube were restreaked, from the relevant plate of the duplicate library, onto L-agar plates containing tetracycline, which were incubated at 37° C. for about 16 hours. A loopful of cells from each restreak was tested for carbamoylase activity using the phenol red test described above. One of the 25 colonies produced a pinkish-red colour. The reaction tube was centrifuged at 12000 x g for 2 minutes to pellet the cells and the supernatant was assayed by HPLC to confirm that D(-)HPG was present. The clone encoding carbamoylase activity was deposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland on 26th Apr., 1989 under the accession number NCIMB 40133. The deposit was made under the Budapest Treaty on the deposition of micro-organisms for the purposes of patent procedure.

1. customCells from half of one plate (i.e. from 25 different clones) of the library were scraped off into a microcentrifuge tube
2. additioncontaining 800 μl of assay buffer
3. additionThe contents of the tube were mixed