• Journal of Microbiology and Biotechnology
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• v.2 no.1
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• pp.35-40
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• 1992

The influence of the nature of plasmids on fermentation parameters such as cell growth, cell viability, plasmid stability, and product formation has been investigated using E. coli M5248 and its recombinant derivatives M5248 [pBR322], M5248[pAS1], and M5248[pNKM21]. At a low temperature ($30^\circ{C}$), the cell growth, cell viability, and protein synthesis of the recombinants were nearly identical to those of the host cell. However, at high temperature ($42^\circ{C}$), in which transcription from the P_L$ promoter is derepressed, the recombinant cells showed decreased stability along with lower growth rates and cell viability. The ratio of total protein to cell mass was in the order of E. coli M5248>M5248[pBR322]>M5248[pAS1]>M5248[pNKM21]. It was found that transcription from the $P_L$ promoter adversely affect the plasmid maintenance and host cell metabolism even in the absence of the cloned-gene expression. Furthermore, profiles of ${\beta}$ activity were shown to vary with recombinant strains. E coli M5248[pBR322] showed highest ${\beta}-lactamase$ activity at $30^\circ{C}$, while at $42^\circ{C}\;{\beta}-lactamase$ activity was significantly reduced irrespective of the strains. The effect of the plasmid properties on plasmid-encoded gene expression has been further examined based on the relationship between $\{beta}-lactamase$ activity and plasmid-harboring cell numbers.

• KSBB Journal
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• v.5 no.3
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• pp.293-298
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• 1990

Alpha-interferon was produced by using recombinant Escherichia coli strains, which carry cloned alpha-interferon gene in plasmid vectors, pIF-III-B and pIF-III-C. With the aid of signal sequence of E. coli lipoprotein, which is placed right in front of the upstream of the cloned alpha-interferon gene of the plasmids, about 50% of alpha-interferon produced was excreted or secreted. Meanwhile, there was no extracellular production of alpha-interferon from the recombinant strain carrying the plasmid Hif-2h that lacks the signal sequence of lipoprotein.

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.35-40
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• 1990

The restriction clevage map of multi-copy recombinant plasmid, pJY712(8.1kb), carrying the thiostrepton resistance gene(tsr) was determined. pJY712 had a broad host range in Streptomyces and contained single BglII site for cloning purpose. The plasmid showed the phenomenon of lethal zygosis ($Ltz^{+}$). Transformation frequency of pJY712 was $5.0\times 10^{4}$ transformants per ug plasmid DNA (TFU) in S. lividans. Plasmid pJY713 was constructed by inserting the tyrosinase gene(mel) into the BclI site of pJY712. Recombinant plasmid pJY714 carrying the mel gene was constructed by in vitro deletion of a segment (1.9kb BglII-BclI fragment) from pJY713.

• Journal of Microbiology
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• v.33 no.3
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• pp.178-183
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• 1995

The recombinant plasmid of pCU103 constructed by cloning pcbCD genes in pBluescript SK(+) was studied for the effect of temperature on its persistence in different waters by the methods of electrophoresis, Southern hybridization, quantification, and transformation. The plasmid was very rapidly degraded out in non-sterile FW water without regards to water temperature, probably due to the effect of biochemical factor such as nucleases. The pCU103 was most persistent at 4$^{\circ}C$ in any water environments, moderately persistant at 15$^{\circ}C$ but least stable at 3$0^{\circ}C$ such results could be explained by the facts that hydrogen bonds in double-stranded plasmid DNAs become unstable and that nucleases are activated by increasing temperature. The intact structure of pCU1-3 was generally observed by gel electrophoresis under the conditions which the plasmid should be 2.0 ng/$\mu\textrm{l}$ or higher in concentration and that about 10$^2$ CFU/ml or more transformant cells should be recovered.

• Journal of Microbiology and Biotechnology
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• v.2 no.2
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• pp.129-134
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• 1992

Segregational instability of a recombinant plasmid, pDML6, encoding extracellular $\beta$-lactamase in Streptomyces lividans PD6 was characterized by growth kinetic analysis. The quantitative determination of the plasmid harbored in the mycelia was evaluated with mycelia fragmented mechanically, and also with colonies regenerated from protoplasts. Conditions for the formation of protoplasts and regeneration of protoplasts were established. The maximal specific growth rates of the host strain and the plasmid-harboring strain in a chemically defined medium without selection pressure were the same. The probability of plasmid loss from the harbouring cells was higher at higher growth rates. Mathematical models for the prediction of cell growth, substrate uptake, and accumulation of the cloned gene product were developed.

• KSBB Journal
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• v.6 no.1
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• pp.9-14
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• 1991

We studied the production and excretion of alpha-interferon in recombinant Escherichia coli harboring plasmid pIF-III-B, which carries alpha-interferon gene under the control of lipoprotein and lacUV5 promoter, and lac operator. Basically, the effects of concentrations of ampicillin and an inducer, IPTG, for the expression of the cloned gene, on the productions of alpha-interferon and plasmid stability were studied. The highest production of alpha-interferon was observed at 50 mg/1 of ampicillin concentration and 0.5 mM of IPTG. The plasmid pIF-III-B was maintained very stably in medium with ampicillin but segregated rapidly in medium without ampicillin. Also, the plasmid was segregated more rapidly in medium with an inducer higher than 0.5 mM.

• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.18-22
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• 1994

Enterococcus faecalis KBL703 has three plasmids(p703/9, p703/5 and p703/4). Within p703/5, the specific DNA region that would confer replication function(replication origin) was searched by transformation experiments. In order to use as the recipient of transformation, two plasmid-cured strainsd were made from this strain. Four recombinant DNA constructs, each containing fragment of p703/5 and CAT(chloramphenicol acetyl transferase) gene were also made. And they were used to transform the plasmid-cured strains. Only one DNA construct containing 3.6 kb SalI fragment was stably maintained as plasmid in these strains. Additional experiment using another Enterococcus faecalis strain(ATCC29212) as a recipient was successfully done and it was confirmed that this newly constructed recombinant plasmid plasimid contained the replication origin from p703/5 plamid.

• Korean Journal of Microbiology
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• v.28 no.3
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• pp.181-187
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• 1990

STA gene coding glucoamylase was introduced into haploid Saccharomyces cerevisiae SHY3 and polyploid Saccharomyces cerevisiae 54. We constructed the recombinant plasmid by substituting the promoter region of alcohol dehydrogenase isoenzyme I gene for that of STA gene to increase the expression of STA gene and found that the activity of glucoamylase was increased in transformants. The plasmid stability was improved remarkably when we got the STA gene into the plasmid which had centromere. The activity of glucoamylase and transformation frequency of it, however, was decreased because of low copy number. Industrial polyploid strain was transformed with the recombinant plasmid having the $2\mu$ origin of replication and STA gene. It produced more alcohol than host when fermented in liquefied starch media. The industrial strain, however, was not transformed with the autonomously replicating plasmid containing centromere.

• Microbiology and Biotechnology Letters
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• v.18 no.1
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• pp.94-97
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• 1990

The restriction cleavage map of multi-copy recombinant plasmid, pJY502 (5.5 kb), carrying the thiostrepton resistance gene (tsr) was determined. Comparison of the restriction pattern with that of Streptomyces plasmids previously demonstrated that pJY502 was novel. The plasmid pJY502 had a broad host range in Streptomyces and contained single BgtII site for cloning purpose. Transformation frequency of pJY502 was $2.2 \times 10^5$ in S. lividans. E. coti-Streptomyces bifunctional plasmid, pJY504, was also constructed.

• Journal of Microbiology and Biotechnology
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• v.2 no.3
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• pp.161-165
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• 1992

The gene encoding the bacteriolytic enzyme cell wall peptidoglycan hydrolase from alkalophilic Bacillus sp. was cloned in E. coli using pBR322 as a vector. A recombinant plasmid, designated pYTR451, was isolated and the size of the cloned HindIII fragment was found to be 4.8 Kb. The cell wall hydrolysis activity of an extract of the E. coli harboring the recombinant plasmid pYTR 451 was detected by SDS- polyacrylamide gel containing 0.2% (w/v) purified cell wall of Bacillus sp. The molecular weight of the enzyme was estimated to be about 27, 000 corresponding to the molecular weight of the Bacillus sp. bacteriolytic enzyme. The recombinant plasmid was found to contain the fragment originated from Bacillus sp. YJ-451 chromosomal DNA by Southern hybridization.