• Applied Microscopy
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• v.20 no.2
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• pp.57-70
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• 1990

Characterization and electron microscopic visualization of the plasmid and the gene expression of Escherichia coli were carried out. Transcriptional units of active structural genes were observed after lysis of Escherichia coli cells. The ribosomes attached to the E. coli genome on mRNA molecule as polyribosomes. From this gradient of polyribosome length, we estimated location of mRNA synthesis initiation site. In this experiment, a granule is ofen present which may correspond to a RNA polymerase at the promoter site. pOX1, pOX7, pOX7A, $pOX7{\Delta}1$, pSTP36, pSTP21, pBR322, and pJH12 were visualized by way of electron microscope, and their estimated sizes were determined to be $5.70{\pm}0.08{\mu}m,\;2.15{\pm}0.10{\mu}m,\;2.14{\pm}0.12{\mu}m,\;7.39{\pm}0.08{\mu}m,\;4.03{\pm}0.04{\mu}m,\;1.50{\pm}0.03{\mu}m\;and\;1.25{\pm}0.09{\mu}m$ respectively. One micrometer of measured length corresponded to about 3.0 Kb. Mica-press adsorption method that allows selectivs visualization of the plasmid DNA released in situ from the bacterial cell is rapid and useful for visualization of plasmids. The released plasmid DNA was adsorbed preferently on mica in a divalent cation-free solution. Miller chromatin-spreading method was useful to observe the plasmid and transcripts. BAC method and cytochrome C monolayer were useful to observe the plasmid DNA. Our ability to visualize ultrastructural aspects of the expression of E. coli has given us a unique tool with which to study the regulation the level of an individual gene.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1357-1363
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• 2008

The $P_{entC}$ promoter of the entCERA operon encoding enzymes for enterobactin biosynthesis in Escherichia coli is tightly regulated by the availability of iron in the culture medium. In iron-rich conditions, the $P_{entC}$ promoter activity is strongly repressed by the global transcription regulator Fur (ferric uptake regulator), which complexes with ferrous ions and binds to the Fur box 19-bp inverted repeat. In this study, we have constructed the expression vector pOS2 containing the $P_{entC}$ promoter and characterized its repression, induction, and modulation by quantifying the expression of the lacZ reporter gene encoding $\beta$-galactosidase. $\beta$-Galactosidase activities of E. coli transformants harboring pOS2-lacZ were highly induced in the presence of divalent metal ion chelators such as 2,2'-dipyridyl and EDTA, and were strongly repressed in the presence of excess iron. It was also shown that the basal level $\beta$-galactosidase expression by the $P_{entC}$ promoter was drastically decreased by incorporating the fur gene into the expression vector. Since the newly developed iron chelator-inducible expression system is efficient and cost-effective, it has wide applications in recombinant protein production.

• Microbiology and Biotechnology Letters
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• v.23 no.3
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• pp.275-281
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• 1995

From the plasmid pYA300 carring a CMCase of Rhizobium fredii USDA193 plasmid was subcloned into pBluescript II KS(+)/pBluescript II SK(+) vectors and designated pYA500 and pYA600, respectively. Escherchia coli cells transformed with pYA500 porduced the CMCase more than with pYA600. The orientation of the cloned fragment in pBluescript vector had the effect on gene expression in E. coli background. When the 1.7 kb CMCase gene fragment of R. fredii USDA193 was hybridized to EcoRI-digested total DNA from R. meliloti and R. fredii USDA 191 the unique bands hybridized respectively, indicating that some genetic diversity exists in the EcoRI restriction enzyme site for CMCase gene in Rhizobium strains. The optimum pH of enzyme activity was 7 and the optimum temperature of that was nearly 37$\circ$C. The cellulase-minus derivatives of pYA500 were constructed by Tn5 insertional mutation. Among 6000 transconjugants, two mutant plasmids (designated pYA500::Tn5a and pYA500::Tn5b) were detected from the cellulase- negative transconjugants. The product of CMCase gene was analyzed by one dimensional SDS- PAGE of the cell extracts. About 45 kDa protein was considered to be a product of CMCase gene.

• Journal of Plant Biotechnology
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• v.8 no.1
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• pp.9-14
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• 2006

As an alternative method to produce low cost reagents for immunodiagnosis and protect the plants from viral disease, a gene encoding a single chain variable fragment(scFv) recombinant antibody targeted to the coat protein of cucumber mosaic virus (CMV) was expressed in Nacotiana tabacum. The source of the scFv recombinant antibody gene was from spleen tissue of an immunized mouse. The gene was initially cloned into the pCANTAB5E phagemid and expressed in E. coli. In the following study, the antibody gene was subcloned into the plant expression vector, pCAMBIA-1301 and introduced into tobacco leaf tissue via Agrobacterium tumefacients mediated transformation. After transformation, 56 out of 58 plants were shown to carry the desired anti-CMV scFv gene by PCR analysis. Overall, only 12.5% of the 56 putative transgenic plants were found to express the antibody to a detectable level.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.316-319
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• 1988

Pectate Lyase (PL) was cloned from alkali-tolerant Bacillus sp. YA-14 into Escherichia coli MB1000 by inserting HindIII-generated DNA fragment into the HindIII site of pBR322 and then screening recombinant transformant for the ability to hydrolyze sodium polypectate on agar plate, The recombinant plasmid, called pYPC29, was isolated, and the size of the cloned HindIII fragment was found to be 1.6 kb. The PL gene was stablely maintained and expressed efficiently in Escherichia coli. The Pt accumulated largely in the periplasmic space of Escherichia coli clones.

• Journal of Microbiology and Biotechnology
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• v.29 no.6
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• pp.944-951
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• 2019

Lipases are industrial enzymes that catalyze both triglyceride hydrolysis and ester synthesis. The overexpression of lipase genes is considered one of the best approaches to increase the enzymatic production for industrial applications. Subfamily I.2. lipases require a chaperone or foldase in order to become a fully-activated enzyme. The goal of this research was to isolate, clone, and co-express genes that encode lipase and foldase from Burkholderia territorii GP3, a lipolytic bacterial isolate obtained from Mount Papandayan soil via growth on Soil Extract Rhodamine Agar. Genes that encode for lipase (lipBT) and foldase (lifBT) were successfully cloned from this isolate and co-expressed in the E. coli BL21 background. The highest expression was shown in E. coli BL21 (DE3) pLysS, using pET15b expression vector. LipBT was particulary unique as it showed highest activity with optimum temperature of $80^{\circ}C$ at pH 11.0. The optimum substrate for enzyme activity was $C_{10}$, which is highly stable in methanol solvent. The enzyme was strongly activated by $Ca^{2+}$, $Mg^{2+}$, and strongly inhibited by $Fe^{2+}$ and $Zn^{2+}$. In addition, the enzyme was stable and compatible in non-ionic surfactant, and was strongly incompatible in ionic surfactant.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.241-247
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• 2005

A new lactococcal shuttle/expression vector for lactococci, pWgal13T, was constructed using a $\beta$-galactosi-dase gene (lacZ) from Lacfococcus lactis ssp. lactis ATCC 7962 as a screening marker. The pWgal 13T was introduced into Escherichia coli DH5a and L. lactis MG1363, and was easily detected by the formation of blue colonies on a medium containing X-gal without any false transformants. Also, the quantitatively lacZ activity of pWgal13T was measured in L. lactis ssp. cremoris MG1363, and was found to be four times higher than that of L. lactis ssp. lactis ATCC7962 grown on a medium containing glucose, which shows that the lacZ gene of pWgal13T can be used for the efficient screening of L. lactis on general media. The pWgal13T was equipped with a lactococcal replicon of pWV01 from L. lactis Wg2, the new promoter P13C from L. lactis ssp. cremoris LM0230, multiple cloning sites, and a terminator for the expression of a relevant gene. The vee-tor pWgal13T was used for the expression of the EGFP gene in E. coli and L. lactis. These results show that the lactococcal expression/shuttle vector constructed in the present study can be used for the production of foreign proteins in E. coli and L. lactis.

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.1
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• pp.6-10
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• 1998

A plasmid vector containing two reporter genes, mer-lux and lac-GFP, was transformed to both Escherichia coli and Pseudomonas putida. Their cellular activities and biofilm characteristics were investigated in flow-cell units by measuring bioluminescent lights and fluorescent levels of GFP. Bioluminescence was effective to monitor temporal cell activities, whereas fluorescent level of GFP was useful to indicate the overall cell activities during biofilm development. The light production rates of E. coli and P. putida cultures were dependent upon concentrations of HgCl2. Mercury molecules entrapped in P. putida biofilms were hardly washed out in comparison with those in E. coli biofilms, indicating that P. putida biofilms may have higher affinity to mercury molecules than E. coli biofilms. It was observed that P. putida expressed GFP cDNA in biofilms but not in liquid cultures. This may indicate that the genetic mechanisms of P. putida were favorably altered in biofilm conditions to make a foreign gene expression possible.

• Journal of Microbiology and Biotechnology
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• v.23 no.2
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• pp.149-155
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• 2013

Heterologous expression of a putative $K^+/H^+$ antiporter of Streptomyces coelicolor A3(2) (designated as sha4) in E. coli and Streptomyces hygroscopicus JCM4427 showed enhanced tolerance to $K^+$ stress, acidic-pH shock, and/or geldanamycin production under $K^+$ stress. In a series of $K^+$ extrusion experiments with sha4-carrying E. coli deficient in the $K^+/H^+$ antiporter, a restoration of impaired $K^+$ extrusion activity was observed. Based on this, it was concluded that sha4 was a true $K^+/H^+$ antiporter. In different sets of experiments, the sha4-carrying E. coli showed significantly improved tolerances to $K^+$ stresses and acidic-pH shock, whereas sha4-carrying S. hygroscopicus showed an improvement in $K^+$ stress tolerance only. The sha4-carrying S. hygroscopicus showed much higher geldanamycin productivity than the control under $K^+$ stress condition. In another set of experiments with a production medium, the secretion of geldanamycin was also significantly enhanced by the expression of sha4.

• Journal of Life Science
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• v.9 no.1
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• pp.54-57
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• 1999

We have got several clones from Serratia marcescens which stimulated the cells to use maltose as a carbon source in E. coli TP2139 (${\Delta}$lac, ${\Delta}$crp). One of the cloned genes, pCKB13, was further analyzed. In order to find whether the increased expression of the gene under the direction of maltose metabolism, we constructed several recombinant subclones. We have confirmed that the clone, pCKB13 codes Coenzyme A transferase gene by partial nucleotide sequencing in the terminal region. The enzyme activity of Coenzyme A transferase increased after introduction of the multicopy of the cloned gene in E. coli. The recombinant proteins expressed by multicopy and induction with IPTG, two polypeptide of 26-and 28-kDa, were confirmed by SDS-PAGE. Southern hybridization analysis confirmed that the cloned DNA fragment was originated from S. marcescens chromosomal DNA.