Restriction digests of the clone give the following sizes (kb): BamHI/EcoRI--3.0, 1.2; PstI--4.1; PvuII--2.5, 1.0, 0.6. Culture distributed in quantities of 0.4 ml in liquid nitrogen vials. Plasmid is not stable when freeze dried. The vector can be used for integration of E. coli alleles at the corresponding locus in the host chromosome by cloning an allele of interest, containing a selectable marker, into MCSII. The plasmid can then be transformed into a recA+ host, selecting for the cloned marker in the presence of p-Cl-Phe (in media lacking tryptone and ampicillin). The host strain DH5alpha is not recommended for preparation of the vector due to the slow growth of transformed cells. High efficiency positive selection cloning vector encoding a gene for dominant sensitivity to p-chloro-phenylalanine (No. 13,071-0, Aldrich, Milwaukee, WI). The Gly294 mutant pheS gene is protected by a patent from Hoechst (Frankfurt, Germany). Recombinant plasmids (containing cloned inserts that interrupt or replace the pheS gene) can be selected for by growth on rich media lacking tryptone and supplemented with 10 mM D,L-p-chloro-phenylalanine and 200 ug/ml ampicillin. (The recommended media is YEG-Cl described in the reference below.) The Ala294-->Gly mutant pheS gene encoded in the plasmid produces an enzyme with relaxed substrate specificity, and is dominantly lethal over the chromosomal pheS gene in the presence of p-Cl-Phe. |
