Abstract:
For high level expression high copy number plasmids are used for cloning purposes. But hybrid plasmids face instabilities due to high level expression or metabolic burdon imposed on hotcells. Hasnain and Thomas (1986) has constructed a novel gene bank of B.subtilis in low copy number vector plasmid, its copy number can be increased under particular conditions.
Plasmids PSH96, PSH263, PSH550, PSH1012 and PSH1019 of this gene bank strongly hybridize with pyr DNA cloned by Chi et al (1978). PSH1012 is a stable plasmid both in low as well as high copy number strains, and it complements pyr gene in Escherchia coil. While other plasmids are not stable when placed at high copy number and undergo various changes. PSH263 and PSH1019 have large inserts and cannot be transformed to high copy number strain. Transformation efficiency of PSH96 and PSH550 is affected severely when high copy strain is used for transformation. Restriction pattern of all these hybrid plasmids, with EcoRl, sa1I, Hind III, Sst II, Pst I has been worked out. Genetic mapping of Pyr loci on the B.subtilis fragment was done by transforming these plasmids to different auxotrops of Pyr loci. PyrD was found to be present in all. Absence of PyrA in PSH263 and PSH1019 may be responsible for instability. Extreme pH do effect plasmid stability. Acidic pH results in insertions or multimer formation while alkaline pH (8 and 9) caused deletions in plasmid DNA. At elevated copy number and with the larger inserts, non-optimal pH had worse effects. All of these plasmids exhibit segregational instabilities when grown in the absence of selection pressure. Initial decrease in the plasmid bearing cells in generally followed by the increase in fraction of plasmid-containing segregants. PSH96 and PSH550 showed more segregational instabilities while PSH1019 was comparatively more stable. pH, temperature and glucose concentration of the medium obviously affect plasmid stability. Any depart from optimum of these factors (Ph7, 350c, 0.2% glucose) results in increase in segregational instabilities of thses plasmids. Increase in copy number under pH or temperature stresses worstly affected plasmid stability, while in varing glucose concentrations of media elevated copy number manifested more plasmid stability. Decrease in Km concentration of the culture medium resulted in segregational loss of hybrid plasmid. Concentration above optimum (50µg/ml) did not exhibit remarkable increase or decrease in plasmid bearing cells. At no or very high selection pressure plasmids undergo structural insatbilities. recA mutation drastically affected segregational stability of hybrid plasmids and fluctuations were also so inconsistent that importance of recombination repair system to stable maintenance of plasmid become evident.
