Inactivation and complementation of microbial genes is commonly done in order to understand gene function. Complementation procedures often utilize plasmids as cloning hosts. Cloning of genes into vector plasmids is, however, often hampered by instability of plasmid isolates due to degradation by copurified DNAases from some bacterial host cells. The objective of this study was to evaluate different methods for overcoming pMMB66EH vector spontaneous degradation and hence facilitate its use for gene complementation studies. HB101 E. coli carrying pMMB66EH were grown in LB prior to plasmid extraction. Extracted plasmids were subjected to different treatments such as heating, phenol purification and switching host cells. Results indicated that pMMB66EH plasmids degraded when exposed to different enzyme buffers or EcoRI alone. The plasmids equally degraded following heating at 65oC or 95oC and after phenol extraction. Obtention of stable pMMB66EH vector for cloning was only possible after switching E. coli hosts. In conclusion it was confirmed that transformation of pMMB66EH from the endA+ host strain (HB101) to endA- host E. coli is an effective procedure for overcoming plasmid spontaneous degradation and hence obtain stable templates for cloning genes.
You may also start an advanced similarity search for this article.