• Korean Journal of Microbiology
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• v.21 no.2
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• pp.95-102
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• 1983

The penicillin G acylase(pga) gene was cloned in the vector plasmid pKM $300(Ar^r,\;Tc^r,\;6.33kb)$ for the study of the structure and expression of the pga gene. This recombinant plasmid pPAKS-1 DNA(24.5 Kb) was cleaved into 2 fragments by restriction enzyme Eco R1.1fragment by BamH1, 4fragments by Hind III, and 2 fragments by Pst I. The pga gene was located on the Eco R1.Hind III-C fragement of pPAKS-1. The recombinant plasmids pPAKS-1 and pPAKS-2, in which the Hind III-B and Hind III-D fragments pPAKS-1 are deleted, are characterized. The results are summarized as follows : 1. Doubling times of bacterial strain bearing pPAKS-1 and pPAKS-2 are 90 and 60 minutes, respectively. 2. pPAKS-1 and pPAKS-2 are present at about 16-32 and 70 copies per cell, respectively, are 0.66 and 5.5 units, respectively, which represent 2-fold and 20-fold higher enzyme 4. pPAKS-1 is very unstable, but pPAKS-2 is stable.

• KSBB Journal
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• v.6 no.3
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• pp.223-230
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• 1991

Using the shuttle vector pECCGl between Escherichia coli and Corynebacterium glutamicum and C. glutamicum strain JS231 grown in the medium supplemented with penicillin-G, which inhibits the formation of cross-links in the peptidoglycan of bacterial cell wall, various parameters involved in electroporation system including resistance, electric field strength, capacitance, DNA concentration, and cell density were investigated independently and optimized for the high efficiency transformation of coryneform bacteria. Using cells grown with 0.3U/ml of penicillin-G and harvested at A600 of 0.7-0.8, transformation efficiencies of 107-l08 transformants/$\mu\textrm{g}$ of DNA with Corynebcctertum glutamicum strain JS231 and wild type ATCC13032 were achieved under conditions of 12.5kV/cm of electric field strength, 400 ohms of resistance, $25\mu$F of capacitance, 3$\times$108 cells per transformation(1.2$\times$1010 cells/ml) and 100ng of plasmid DNA per transformation.

• BMB Reports
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• v.31 no.3
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• pp.240-244
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• 1998

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a major virulence factor and in a potent immunogen. In order to express the recombinant urease at a higher level, a DNA fragment containing the minimal H. pylori urease gene cluster was subcloned into a high copy-number vector. The recombinant H. pylori urease expressed in an E. coli strain that was grown in a rich medium supplemented with added nickel was purified to near homogeneity by using DEAE-Sepharose, Superdex HR200, and Mono-Q (FPLC) columns and the purified enzyme possessed the specific activity of 1255 U/mg. Polyclonal antibodies raised against the purified recombinant H. pylori urease were shown to be very specific when subjected to Western blot analysis, in which crude extracts from the H. pylori ATCC strain and the recombinant E. coli strains expressing various bacterial ureases were exnmined for cross-reactivity.

• Microbiology and Biotechnology Letters
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• v.23 no.1
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• pp.104-109
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• 1995

The gene responsible for dechlorination of 4-chlorobenzoate (4CBA) was cloned in E. coli XL1-Blue from Pseudomonas sp. DJ-12. The cloned cell of E. coli Cjl had the hybrid pBluescript SK(+) plasmid, into which about 9.5 kb genomic DNA fragment of PseudOmonas sp. DJ-12 was inserted. The subclone of pCJlOl was constructed by inserting the 3.4 kb EcoRI-HindIII fragment of pCJl into the vector. Those cloned cells could be simply selected by halo formation around the colonies which was the precipitate of AgCl produced by reaction of AgNO$_{3}$ and chloride ion liberated by bacterial dechlorination of 4CBA- Such a plate assay method was standardized by the procedure that the colonies grown for 2 days on the Cl$^{-}$-free plate medium containing 1 mM 4CBA were flooded with 0.1 M AgNO$_{3}$ solution.

• Journal of Microbiology and Biotechnology
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• v.5 no.2
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• pp.87-91
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• 1995

The chitinase gene was cloned from Acinetobacter sp. WC-17 for investigating the genetic control and enzymatic properties of bacterial chitinase. A genomic library of Acinetobacter sp. WC-17 was prepared in E.coli JM109 by using pUC18 as a vector. The chitinase-positive clone containing 3.2kb insert fragment was obtained from 5, 000 insert-bearing transformants. The optimum pH and temperature of cloned enzyme were 6.0 and $55^{\circ}C$, respectively. Almost all the chitinase activity of E.coli recombinant was localized in the periplasmic fraction, while most of the enzyme activity of Acinetobacter sp. WC-17 was found in the extracellular fraction.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.331.3-332
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• 2002

A novel penicillin G acylase (PGA)-producing bacterial strain was isolated from soil by using the Serratia marcescens overlay technique. The isolated strain was identified as Leclercia adecarboxylata based on the analyses of the biochemical characteristics (API 20E). the cellular fatty acid profile. and the 16S rDNA sequences. The gene encoding the PGA (pac gene) was cloned into the pHSG399 vector and the recombinant E. coliHB101 clones harboring the pac gene were isolated on agar plates containing phenylacetyl-L -leucine and penicillin G. (omitted)

• Archives of Pharmacal Research
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• v.12 no.3
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• pp.176-180
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• 1989

Four bacterial strains having inducible resistance to erythromycin were isolated from soil samples in Korea and characterized. MLS inducibility was checked in each strain. Cloning of inducible resistance gene(s) has been tried. Four isolates were identified as B. anthracis, Micrococcus luteus, Streptococcus faecium and B. licheniformis, in which erythromycin, oleandomycin, cirramycin and carbomycin acted as resistance inducers respectively. The resistance gene cloned from B, licheniformis 597 strain using pBS 42 vector was found to have a 3.2 kb insert.

• Fisheries and Aquatic Sciences
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• v.6 no.1
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• pp.20-26
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• 2003

A bacterial strain was isolated from seawater to screen for phytase activities. A colony had the highest activity and was identified as an Escherichia coli strain. Using primers derived from E. coli acid phosphatase appA sequence, we cloned a 1,495 bp DNA fragment connected with the pGEM-T vector. It was over-expressed under lac promoter combined with its native promoter in E. coli $DH5\alpha$. The expression of the phytase gene occurred during late exponential growth and the intracellular phytase production was 16.9 units/ml. The yield of recombinant phytate was 412-fold higher than that of wild type E. coli K13.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.11a
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• pp.165-169
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• 1996

The cytochrome P450 (P450) proteins have been studied extensively because of their prominent roles as catalysts in the oxidations of drugs, carcinogens, steroids, alkaloids, vitamins, and other important chemicals (Guengerich, 1991). In the past decade the study of these enzymes has been advanced by the cloning of cDNAs and expression of the proteins in several heterologous vector systems. One approach that has been employed in this and other laboratories is expression in bacteria. To date at least 31 different mammalian P450s have been expressed in Escherichia coli-band systems (Guengerich et al., 1996). In most of these cases the N-terminus has been altered to facilitate better expression (Barnes et al., 1991).

• Mycobiology
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• v.29 no.3
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• pp.132-134
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• 2001

Since it is known that Agrobacterium tumefaciens, which has long been used to transform plants, can transfer the T-DNA to yeast Saccharomyces cerevisiae during tumourigenesis, a variety of fungi were subjected to transformation to improve their transformation frequency. In this study, I report the A. tumefaciens-mediated transformation of filamentous fungus Aspergillus niger. Transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator as a selectable marker, led to the selection of $50{\sim}100$ hygromycin B-resistant transformants per $1{\times}10^7$ conidia of A. niger. This efficiency is improved $10{\sim}20$ fold more than reported elsewhere. In order to avoid the difficulties in selection transformant from the over-growing non-transformant, I used top agar containing 900 ${\mu}g/ml$ of hygromycin. Genomic PCR and Southern analysis showed that all transformants contained single T-DNA insert per fungal genome. This technique offers an easier and more efficient method than that of using protoplast.