Analysis of the instability of trp operon recombinant plasmid in Escherichia coli

Abstract

Mathematical analysis method of recombinant plasmid instability was devised and applied to practical quatitization of the factors involved in plasmid instability. As a model system, E. coli trp opron recombinant strain MV12/pVH5 (Table 1) was chosen and host repression system was partially derepressed by various concentration of indoleacrylic acid. The result from kinetic test of plasmid instability showed that the stability of pVH5 and the growth rate of MV12/pVH5 were rapidly decreased at above 10 ug/ml of indoleacrylic acid but decreased no more over 40 ug/ml. These suggested that host cell produced trp- variants from MV12/pVH5 at different frequency depending on physiological condition and has some mechanism on the plasmid modification. In parallel, MV12trpR/pVH5 strain was constructed by conjugation of MV12/pVH5 and CSH61 and the stability of pVH5 was assayed also. The stability of pVH5 in MV12trpR mutant was greatly decreased compared to that by the effect of indoleacrylic acid but the result of plasmid loss kinetics test was consistent with above description. The cleared lysates prepared from $trp^-$ variants mostly had modified plasmids of increased sizes, indicating plasmid modification was the main mechanism of plasmid instability. As the gene source of trp operon, transducing phage $Φ80h^-$ pttrp190 or recombinant plasmid pVH5 and as a cloning vehicle pBR322 were used and both were partially digested with restriction enzyme EcoRI and covalently linked with T4 ligase. The ligation mixture was transformed into E. coli $JA221r_K^-m_K^+$ recAΔtrpE5 and MV12(C600 $r_K^-m_K^+$ recAΔtrpE5), then recombinants were isolated on VB minimal plate containing ampicillin 50 ug/ml and tetracyclin 10 ug/ml. Interestingly, among the recombinants of JA221/pBR322-trp, one harbored an additional pVH5 plasmid simultaneously, and the stability of pBR322-trp in this strain was somewhat lower than that in the JA221 harboring pBR322-trp only.