• Microbiology and Biotechnology Letters
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• v.15 no.6
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• pp.396-401
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• 1987

Protein productivities of a cloned gene ($\beta$-galactosidase) and the cultural performances of two recombinant Escherichia coli strains, which use different host-vector systems, were studied. E. coli JM109/pTBG10 strain which carries Tac promoter had higher protein productivity than E. coli MH3000 (pRKc1857)/pASI(lacZ) strain which carries pL promoter. Induction of protein syn-thesis was optimum at the initial-and mid-logarithmic growth phases for both strains. Oxygen demand was observed to be very high during the cloned gene expression, and could be alleviated to some extent through pH control. The ratio of specific growth rates of plasmid-harboring to plasmidfree cell, $\mu$+ /$\mu$-, of the high productivity strain was observed to be lower than that of the low productivity one. Plasmid stability was analyzed for 20-30 generations, and it was found that the traction of plasmid-harboring cells dropped to l0% level in about 25 generations for both strains when the cloned gene expression was induced.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.550-553
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• 2003

The genes of GFPuv and Cytochrome c-552 were amplified by using PCR, and then, fused each other. Fusion gene of GFPuv and Cytochrome c-552 was inserted into the pTrcHis B vector and transferred to E. coli. A fusion protein of GFPuv and Cytochrome c-552 was expressed in JM109 and BL21. This fusion protein was composed of a His-tag for the rapid one-step purification using an immobilized metal affinity chromatography.

• Preventive Nutrition and Food Science
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• v.15 no.4
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• pp.360-363
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• 2010

To enhance the expression and secretion of pediocin PA-1 from heterologous bacterial hosts, the promoter and deduced signal sequence (PS) of an $\alpha$-amylase gene from a Bifidobacterium adolescentis strain was fused with pediocin PA-1 structural and immunity genes (AB) and the resulting functions were evaluated in Escherichia coli. Two recombinant PCR products were created-one with just the deduced signal sequence and one with the sequence plus the Ser and Thr sequences that are the next two amino acids of the signal sequence. These two products, the PSAB (---AQA::KYY---) and PSABST (---AQA$\underline{ST}$::KYY---), respectively, were inserted into a TA cloning vector (yT&A) and named pPSAB, which was previously reported, and pPSABST. The two recombinant plasmid DNAs were transferred into E. coli JM109 and the transformants displayed antimicrobial activity, where the activity of E. coli JM109 (pPSAB) was stronger than that of E. coli JM109 (pPSABST), indicating that the ST amino acid residues were not necessary for secretion and might have even decreased the antimicrobial activity of recombinant pediocin PA-1.

• Proceedings of the Korean Society of Life Science Conference
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• 2008.10a
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• pp.68.1-68.1
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• 2008

• Biomedical Science Letters
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• v.5 no.1
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• pp.17-26
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• 1999

To evaluate the spreading possibilities of the marRAB mutation of E. coli Mar mutant among gram-negative bacilli, chromosomal marRAB mutations of Mar mutants were transduced by $\lambda$placMu9 into pUC19 (Lac$^{+}$, Ap$^{r}$) cloning site in another strains of E. coli or onto the chrmosome of S. typhimurium and P. aeruginosa, selected for transduction by Mar phenotype, Lac$^{-}$, or Ap$^{r}$, and tested for their antimicrobial resistance with or without addition of salicylate (SAL). Compared with wild type strains of JM109, NM522, harboring pUC19 or not, respectively, all strains of JM109 or NM522 carrying pUC19::marRAB mutation showed higher levels of antimicrobial resistance and SAL induction of Mar phenotype than those of wild type. However, in contrast to the original Mar mutants, there were some tendencies of decreased antimicrobial resistance of JM109 or NM522 harboring pUC19::marRAB mutation with SAL induction against chlorarnphenicol (Cm) and tetracycline (Tc), or Tc and ciprofloxacin (Cp), respectively. Almost the same results, as shown as the cases of E. coli JM109 or NM522, were obtained from all transductants of S. typhimurium and P. aeruginosa, except Cp, against which increased antimicrobial resistance with SAL induction was shown. This study, employed the methods of transformation or transduction among intercellular gene transfer methods between gram-negative bacteria, shows the evidences that suggest indirectly the spreading possibilities of marRAB mutation among gram-negative bacilli.

• Microbiology and Biotechnology Letters
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• v.20 no.6
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• pp.637-641
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• 1992

The promoter regions from chromosomal DNA of Bacillus sp. SSA3 which is responsible for fermentation of Korean traditional soy sauce, were cloned for construction of expression vector of Bacills sp. SSA3. Recombinant plasmids were constructed by insertion of HindIIl-cleaved Bacillus sp. SSA3 chromosomal DNA fragments in front of the CAT gene of pGR71 plasmid and B-galactosidase gene of pUC18 plasmid. 6 recombinant plasmids were isolated from chloramphenicol resistant E. coli JM109 clones. All these plasmids were found to have promoter activity in Bacills sp. SSA3 and E. coli JM109. When these 6 clones of Bacills sp. SSA3 were cultivated in LB agar medium supplemented with 10% NaCI. fused CAT gene expression of 4 clones was significantly decreased in common. But the others were poorly inhibited.

• Journal of Life Science
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• v.21 no.9
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• pp.1205-1213
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• 2011

The optimal concentrations of salts in medium for cell growth and the production of carboxymethylcellulase (CMCase) by a recombinant E. coli JM109/DL-3 were established using two statistical methods: orthogonal array method (OAM) and response surface method (RSM). The analysis of variance (ANOVA) of data based on OAM indicated that $K_2HPO_4$ gave maximum sum of square (S) and percentage contribution (P) for cell growth as well as production of CMCase. The optimal concentrations of $K_2HPO_4$, NaCl, $MgSO_4{\cdot}7H_2O$, and $(NH_4)_2SO_4$ in medium for cell growth extracted by Qualitek-4 (W32b) Software were 10.0, 1.0, 0.2, and 0.6 g/l, respectively, whereas those for the production of CMCase by E. coli JM109/DL-3 were 5.0, 1.0, 0.4, and 0.6 g/l. The analysis of variance (ANOVA) resulting from RSM indicated that a highly significant salt for cell growth was $K_2HPO_4$ ("probe>F" less than 0.0001), whereas $K_2HPO_4$ and $MgSO_4{\cdot}7H_2O$ were significant for the production of CMCase. The optimal concentrations of $K_2HPO_4$, NaCl, $MgSO_4{\cdot}7H_2O$, and $(NH_4)_2SO_4$ for cell growth extracted by Design Expert Software were 7.44, 1.08, 0.22, and 0.88 g/l, respectively, whereas those for production of CMCase were 5.84, 0.69, 0.28, and 0.54 g/l. The optimal concentrations of salts and their influences on cell growth and production of CMCase extracted by OAM were almost the same as those by RSM. Production of CMCase by a recombinant E. coli JM109/DL-3 under optimized concentration of salts was 1.93 times higher than that by Bacillus amyloliquifaciens DL-3.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.227-231
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• 1991

A gene coding for a $\beta$-galactosidase of Bacillus subtilis HP-4 was cloned in E. coli JM109 by inserting HindIII digested fragment of B. subtilis HP-4 chromosomal DNA into the site of pBR322 and selecting recombinant transformant showing blue color on X-gal plate. The recombinant plasmid, named pBG109, was found to contain the 1.4 Kbp HindIII fragment originated from B. subtilis HP-4 chromosomal DNA by Southern hybridization. The cloned gene was stably maintained and expressed in E. coli JM109 and the pBG109 encoded $\beta$-galactosidase had the same enzymatic properties as those of $\beta$-galactosidase produced by B. subtilis HP-4.

• Microbiology and Biotechnology Letters
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• v.20 no.3
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• pp.280-288
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• 1992

Molecular cloning of metalloprotease gene from Serratia marcescens ATCC 21074 into Escherichia coli JM109 was carried out. Chromosomal DNA of S. marcescens was completely digested with Hind111 and southern hybridization with a synthetic oligonucleotide probe revealed that a 50 KD metalloprotease gene was contained in 4.0 Kb chromosomal DNA fragment, 4.0 Kb chromosomal DNA fragments eluted from agarose gel were ligated with pUC19 and transformed into E. coli JM109. Nine positive clones were obtained from about $1\times 10^3$ transformants by colony hybridization. Their recombinant plasmids, pSPl and pSP2 have same chromosomal DNA fragments in pUC19 in opposite-orientations. When cloned metalloprotease gene was expressed in E. coli, about 52 KD precursor protein of metalloprotease was detected by western blot analysis from E. coli harboring a recombinant plasmid pSP2. Plasmid pSP2 showed no protease activities in E. coli but overproduced the active metalloprotease in S. rnarcescens ATCC 27117.

• KSBB Journal
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• v.24 no.2
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• pp.207-211
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• 2009

In this study, the sensing membranes for detection of pH and dissolved oxygen(DO) were prepared by immobilizing 6-aminofluorescein or ruthenium complex onto the sol-gel matrixes of GPTMS, MTMS, and TEOS and then recoated with the mixture of hydrophobic sol-gel and graphite for light insulation. The pH and DO sensing membranes recoated with the light insulation layer showed a higher sensitivity than those without light insulation layer. The sensing membranes were immobilized on the wells of 24-well microplate and used to monitor the fluorescence intensity for pH and DO in E.coli JM109 and P.pastoris X-33 fermentation processes. The change of the fluorescence intensity in the DO sensing membrane agreed with the growth patterns of microorganisms, that the membranes are valuable to monitor the DO in fermentation processes. In the case of pH monitoring, the fluorescence intensity has showed good correlation to the off-line pH data, that the pH membranes are valuable to monitor pH values in fermentations.