Escherichia coli relA strains as hosts for amplification of pBR322 plasmid DNA

Abstract

We have proposed that guanosine tetraphosphate produced in Escherichia coli cells subjected to an isoleucine limitation inhibits pBR322 DNA replication [1]. In E. coli reiA which cannot synthesize guanosine tetraphosphate (ppGpp) upon amino acid limitation pBR322 DNA is amplified after arginine starvation. The yield of plasmid DNA amplified either by chloramphenicol (Cm) or by arginine limitation is compared. The plasmid yidd per cell is equal in amino acid-starved cells and in cells treated with Cm. To increase the plasmid content per ml of cell suspension the growth medium was supplemented with increasing amounts of nutrients. Plasmid DNA can be isolated in large quantities by this procedure. This simple method can be used for the enrichment of pBR325 DNA which cannot be amplified by Cm treatment. Our results indicate that E. coli relA strains might be suitable hosts for the amplification of pBR322 and related plasmids in E. coli. INTRODUCTIONE. coil [2][3][4]. However, experiments, involving amino acid starvation to inhibit protein synthesis only resulted in a low enrichment of plasmid DNA. In both cases protein synthesis is inhibited but only in amino acid-starved cells ppGpp is synthesized whereas Cm treatment prevents the formation of ppGpp [1,5]. Possibly ppGpp can inhibit plasmid amplification in amino acid-starved cells. Studying this problem we have shown that in E. coli relA strains which cannot produce ppGpp upon amino acid limitation pBR322 DNA is amplified after isoleucine starvation [1]. These resuits support our assumption of a negative control of pBR322 DNA replication by ppGpp.For cloning experiments new methods are constantly being sought to improve the yields of plasmid DNA from cultures of bacterial cells [6,7]. To isolate pBR322 DNA in large amounts, e.g. for recombinant DNA research, the method of plasmid amplification by Cm is widely used [8]. In this study the yields of plasmid DNA amplified either by Cm or by amino acid starvation of the E. coli relA strain CP79 will be compared. Our results indicate that E. coli relA strains might be suitable hosts for amplification of pBR322 plasmid DNA.Inhibition of protein synthesis by Cm causes a rapid amplification of ColEl-related plasmids in 0378-1097/85/$03.30

DNA plasmid production in different host strains of Escherichia coli

“…Hecker et al [18] and Reinikainen et al [34] found that plasmid yields were highest after the culture had entered the stationary phase. Heat treatment, which was accompanied by a reduction in feed rate, resulted in a 50% decrease in growth rate for strains at either growth rate, and therefore a heat treatment may mimic a stationary phase for DNA production.…”

Replication of plasmids derived from P1, F, R1, R6K and RK2 replicons in amino acid‐starved Escherichia coli stringent and relaxed strains

“…Apart from providing basic information about the mechanisms of regulation of DNA replication, studies on replication of plasmids during stringent and relaxed response led to development of a new method for DNA amplification in vivo (HECKER et al 1985). It was found that appropriate amino acid starvation may be used as a mean of achieving effective amplification of plasmids derived from ColE1, pSClOl and bacteriophage Iz (HECKER et al, 1985SCHROETER et al 1988, RIETHDORF et al 1989, HOFMANN et al 1990HER-MAN et al 1994a, b, c, WQGRZYN, 1995, NEUBAUER et al 1996. Our results indicate that using this method it is possible to achieve significant amplification of plasmids derived from P1, F, R1 and R6K replicons.…”

“…Analogous investigations on the stringent control of Bacillus subtilis chromosome replication led to discovery of an interesting general mechanism of replication regulation (SEROR et al 1986, LEVINE et al 1991. Secondly, appropriate amino acid starvation has been reported as an efficient method for in vivo DNA amplification in the case of ColE1, 1, and pSClOl replicons (HECKER et al, 1985, RIETHDORF et al 1989, HOFMANN et al 1990, HER-MAN et al 1994a, b, c, WEGRZYN 1995, NEUBAUER et al 1996. Therefore, we decided to investigate replication of other replicons during stringent and relaxed response in E. coli.…”

DNA degradation at elevated temperatures after plasmid amplification in amino acid-starved Escherichia coli cells

“…Plasmids (the most commonly used vectors in molecular cloning) may be amplified by different methods. Amino acid starvation ofK coii relA strains was proposed as one of methods for amplification of ColEl plasmids (Hecker et al, 1985;Schroeter et al, 1988;Riethdorfet al, 1989;Hofmann et al, 1990). It has been demonstrated that this method may be even more efficient than the commonly used chloramphenicol method (Clewell, 1972;Guzman et al, 1988;Neubauer et al, 1990).…”

“…Recently the conditions for h plasmid DNA amplification during the relaxed response have been optimized (Wegrzyn, 1995). This method of plasmid DNA amplification is very simple and extremely cheap (Hecker et al, 1985;Wegrzyn, 1995). Moreover, it was demonstrated that very efficient overexpression of a gene cloned on a plasmid occurs after simple addition of deprived amino acid following plasmid amplification .…”