Agarose gel analysis of plasmid DNA from sample timepoints from inducible fed-batch fermentation process. A. Agarose gel analysis of gWiz-D plasmid DNA from timepoints during 30°C growth restriction phase (Samples 1–3) 42°C plasmid amplification phase (Samples 4–6) and after holding at 21°C (Sample 7). Arrow indicates dimer and nicked plasmid band that is repaired during hold. B. Two stage agarose gel analysis of pNTCUltra1 RF48 fermentation plasmid DNA samples (see Table I), from end of 42°C induction (1432 mg/L, 14.7 mg/L/OD₆₀₀ specific yield) and after 1 h at 25°C (1560 mg/L, 16.9 mg/L/OD₆₀₀ specific yield). U = untreated plasmid in 1 × DNA gyrase buffer; PL = plasmid was incubated in 1× DNA gyrase buffer with dNTP, E. coli DNA Pol I (P) and T4 DNA ligase (L), for 1 h at 37°C prior to analysis; Exo = plasmid was incubated with T5 exonuclease (digests linear, nicked and denatured DNA) in 1× DNA gyrase buffer for 1 h at 37°C prior to analysis; G = plasmid was incubated 1× DNA gyrase buffer with E. coli DNA gyrase (G), for 1 h at 37°C prior to analysis; PLG = plasmid was incubated in 1× DNA gyrase buffer with dNTP, E. coli DNA Pol I (P), T4 DNA ligase (L) and E. coli DNA gyrase (G)(Topogen, Port Orange, FL) for 1 h at 37°C prior to analysis. OC = open circular nicked plasmid, D = supercoiled dimer plasmid, L = linear plasmid, CCC = covalently closed circular monomer plasmid, relaxed = not supercoiled, super = supercoiled. Note that addition of DNA gyrase (G, PLG lanes) increases negative supercoiling of CCC plasmids, slowing mobility of the monomer and dimer.

A. Inducible fed-batch plasmid fermentation process. Plasmids containing the pUC origin are induced by the 30°C to 42°C shift. B. E. coli strain DH5α containing pNTCUltra1 plasmid inducible fed-batch fermentation growth and plasmid yield profiles. Induction (30°C to 42°C shift) was at 29 h, plasmid yield reached 2130 mg/L.

A. Annotated pNTCUltra-1 restriction map. Critical features such as the trpA terminator, dual terminator site from pNTCUltra1-term+ and functional domains in the origin (PAS-BL, RNAII promoter, RNase H cleavage site) are indicated. B. Plasmid DNA replication steps are shown, with critical gene products indicated. For the leading strand, a RNA polymerase transcript (RNAII) forms an R-loop (RNA DNA hybrid) that is cleaved by RNase H, the resulting 3′ OH serving as a primer for DNA polymerase I (Pol I) DNA synthesis. DNA synthesis then converts to DNA Pol III about 400 bp downstream of the origin. In pBR322, Pol III leading strand synthesis may be initiated by primosomal assembly at either phiX174 type (at PAS-BH, a leading strand primosomal assembly site; Minden and Marians, 1985) or dnaA type (at dnaA binding sites within the origin; Parada and Marians, 1991) assembly sites. PAS-BH is deleted in pUC origin vectors. Replication of the leading strand exposes a phiX174 type primosome assembly site on the lagging strand (PAS-BL), which binds priA, which, in turn, recruits the remaining proteins to form the preprimosome [priB, dnaT, recruits dnaB (delivered by dnaC)], which then also recruits primase (dnaG), which then makes a short RNA substrate for DNA polymerase III. PriC may also be involved in this pathway. Lagging strand replication initiated at PAS-BL terminates at a termination site (ter) within the origin. Leading strand synthesis, with dnaB/dnaC bound to polIII generating lagging strand Okazaki fragments every 1–2 kb, proceeds around the plasmid, also stopping at the ter site. DNA Pol I and DNA ligase are required after Pol III replication to remove primers and seal nicks, respectively. DNA gyrase then negatively supercoils the resultant covalently closed circular (CCC) plasmid. Plasmid replication is different than chromosomal replication, wherein initial replication is set by OriC priming initiated with dnaA (reviewed in Masai and Arai, 1996).