RECOMBINANT PLASMID RETENTION AND EXPRESSION IN BACTERIAL BIOFILM CULTURES

Any exposure of plasmid recombinant microorganisms to an open system environment, either inadvertently or intentionally, mandates research into those fundamental organism:plasmid processes that influence plasmid retention, transfer and expression. In open environmental systems a majority of the microbial activity occurs associated with an interface, within thin biological layers consisting of the cells and their insoluble extracellular polymer, layers known as biofilms. Thus any study regarding the fate of recombinant DNA sequences in an open system muse consider processes that affect plasmid retention and expression in a biofilm culture. Biofilm cultures were cultivated in a parallel-plate flow cell reactor using E. coli DH5 alpha which contained a recombinant plasmid with a plasmid stability factor, parB, (pTKW106) or without (pMJR1750). Using beta-galactosidase as inducible reporter protein, plasmid retention and gene expression of pMJR1750 and pTKW106, in suspended versus biofilm cultures, were studied under different carbon to nitrogen ratios and plasmid induction levels. Recombinant biofilm formation under these environmental conditions was also investigated. Biofilm net accumulation late of E. coli DH5 alpha (pTKW106) decreases with increasing induction levels. The beta-galactosidase production and ratios of beta-galactosidase to total protein increase with increasing induction levels. Synthesis rates of total RNA, beta-galactosidase mRNA and rRNA in biofilm cultures of E. coli DH5 alpha (pTKW106) increase after induction by IPTG.