Effects of the stringent response on pBR322 plasmid copy number in Escherichia coli strains

This experiment attempted to study the replication control of pBR322 during stringent growth in E. coli. If the stringent response is responsible for the control of pBR322 copy number, an amino acid-starved stringent E. coli strain would be expected to decrease plasmid replication, whereas relaxed mutant strains would exhibit amplification of pBR322. In this experiment, the growth rate of an E. coli relA wild-type strain was reduced when the stringent response was stimulated through inhibition of valyl-tRNA synthetase. As expected, the isogenic E. coli relAstrain exhibited similar growth kinetics to the control. However, spectrophotometric quantification, gel electrophoresis band assessment, and a minimum inhibitory concentration assay using tetracycline showed no significant difference in plasmid copy number between the two strains during stringent growth. Both displayed a decrease in pBR322 copy number when deprived for the amino acid valine, leading us to conclude that other mechanisms besides the stringent response are involved in regulating plasmid copy number. _________________________________________________________________