• Korean Journal of Microbiology
• /
• v.42 no.1
• /
• pp.12-18
• /
• 2006

Thirty five Salmonella enterica serovar Typhimurium strains were isolated from diarrheic patients and pigs in Gyeongbuk area from 2003 to 2004. All 35 strains (17 strains from diarrheic patients and 18 from pigs) were resistant to more than one drug and most of strains isolated from pigs were resistant to ampicillin, ohloram-phenicol, streptomycin, sulfamethoxazole-trimethoprime, tetracyclin and nalidixic acid. Each isolate was also screened or the presence of class I, II and III integron gene cassettes. Among 35 strains,3 out of 17 strains isolated from diarrheic patients, carried dhfrX-orfF-aadA2 integron gene cassette and among 18 strains isolated from diseased pigs, 11 strains carried dhfrX-orfF-aadA2 integron gene cassette and 1 strain carried aadA2 integron only. But any class II and class II integron gene cassette were not detected in 35 strains. Thirty five strains were divided by five pulsotypes. Thirty one strains out of thirty five were pulsotype A. Among the remaining 4 strains, one each strain belonged to pulsotype B, C, D and pulsotype E. This data of pulsotypes showed that the widespread of pulsotype A, Salmonella enterica serovar Typhimurium in human and pigs in Gyeongbuk area may have been caused by the dissemination of a few epidemic strains in this area. Thirteen strains contain dhfrX-orfF-aadA2 integron gene cassette showed pulsotype A and one strain contains dhfrX-orfF-aadA2 integron gene cassette showed pulsotype B. One strain contains aadA2 integron showed pulsotype E. But fifteen strains do not contain any integron showed pulsotype A.

• Journal of Life Science
• /
• v.18 no.1
• /
• pp.136-142
• /
• 2008

A recombinant Pichia pastoris carrying double expression cassette of Rhodotorula glutinis epoxide hydrolase(RgEH) gene was developed and used for preparing enantiopure (S)-styrene oxide from racemic mixture of styrene oxide. BglII restriction site of original RgEH gene (pPICZ B/RgEH #2) of previous report was mutated using PCR technique for the construction of double expression cassette containing promoter ($P_{AOX1}$), EH gene and transcription terminator ($TT_{AOX1}$) in pPICZ C vector. Double expression cassette with RgEH was inserted into the chromosomal DNA of P. pastoris. $V_{max}$ ($2.2{\mu}mol\;min^{-1}mg\;dcw^{-1}$) on (R)-styrene oxide of P. pastoris with double expression cassette was about 6-fold higher than that ($0.4{\mu}mol\;min^{-1}mg\;dcw^{-1}$) of P. pastoris with single expression cassette. For the determination of the optimal condition, the effects of detergent and temperature on the enantioselective hydrolytic activity and yield of the enantiomer were investigated. When the reaction was performed at $10^{\circ}C$ for 10 min in the presence of 0.5% Tween 20, enantiopure (S)-styrene oxide with 99.9% ee was obtained as the yield 43.4 % from 20 mM racemic sustrate.

• Korean Journal of Microbiology
• /
• v.41 no.2
• /
• pp.146-151
• /
• 2005

To construct an amylolytic industrial strain of Saccharomyces cerevisiae, the glucoamylase cDNA gene (GAl) from Aspergillus awamori was expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p) and integrated into the chromosomes of industrial S. cerevisiae. An integrative cassette lacking bacterial ampicillin resistance gene but containing the GA1 gene, $\delta$ sequences of Ty1 retrotransposon as target sites for homologous recombination and S. cerevisiae aureobasidin A resistance gene (AUR1-C) as the selection marker was constructed to obtain a strain eligible for commercial use. Industrial S. cerevisiae transformed with this 15-integrative cassette efficiently secreted glucoamylase into the medium and grew on starch as the sole carbon source. The transformants were mitotically stable for 100 generations in nonselective medium.

• Journal of Life Science
• /
• v.28 no.11
• /
• pp.1277-1284
• /
• 2018

In this study, a repeated yeast integrative plasmid (R-YIp) harboring Cre/loxP system was constructed to integrate various gene expression cassettes into the yeast chromosome. The R-YIp system contains a reusable selective marker (CgTRP1), loxP sequence, and target sequence for integration. Therefore, many gene expression cassettes can be integrated into the same position of the same yeast chromosome. In the present study, several model enzymes involving xylan/xylose metabolism were examined, including endoxylanase (XYLP), ${\beta}$-xylosidase (XYLB), xylose reductase (GRE3) and xylitol dehydrogenase (XYL2). Efficient expression of these genes was obtained using two promoters (GAL10p and ADH1p) and various plasmids (pGMF-GENE and pAMF-GENE plasmids) were constructed. The XYLP, XYLB, GRE3, and XYL2 genes were efficiently expressed under the control of the GAL10 promoter. Subsequently, R-YIps containing the GAL10p-GENE-GAL7t cassette were constructed, resulting in pRS-XylP, pRS-XylB, pRS-Gre3, and pRS-Xyl2 plasmids. These plasmids were sequentially integrated into chromosome VII of a Saccharomyces cerevisiae strain by repeated gene integration and selective marker rescue. These genes were integrated by the R-YIp system and were stably expressed in the yeast transformants to produce active recombinant enzymes. Therefore, we expect that the R-YIp system will be able to overcome current limitations of the host cells and allow selective marker selection for the integration of various genes into the yeast chromosome.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.7
• /
• pp.1008-1013
• /
• 2014

The use of antimicrobial agents for additives or therapeutics is strongly associated with a prevalence of antimicrobial resistance in commensal Enterobacteriaceae. We aimed to characterize integrons in Enterobacteriaceae isolates obtained from chicken cecums in Korea. Moreover, the correlation between integron gene cassettes and antimicrobial resistance was also investigated. A total of 90 isolates the belonged to Enterobacteriaceae were recovered from chickens grown at Gyeongsang and Chungcheong provinces in Korea. Antimicrobial susceptibility tests were performed by the disk diffusion method. PCR and DNA sequencing were also performed to characterize the gene cassette arrays of the integrons. Of the 90 Enterobacteriaceae isolates tested, 39 (43.3%) and 10 (11.1%) isolates carried class 1 and 2 integrons, respectively. Whereas the class 2 integron did not contain gene cassettes, the class 1 integrons carried seven different gene cassette arrays. The class 1 integrons harbored genes encoding resistant determinants to aminoglycosides (aadA1, aadA2, and aadA5), trimethoprim (dfrA1, dfrA12, dfrA17, and dfrA32), lincosamides (linF), and erythromycin (ereA). Moreover, the presence of a class 1 integron was significantly related to a high resistance rate of antimicrobial agents, such as spectinomycin and trimethoprim. We confirmed that diverse class 1 integrons were widely distributed in Enterobacteriaceae isolates from chickens and directly contributed to the resistance to diverse antimicrobial agents in Korea.

• Journal of Microbiology and Biotechnology
• /
• v.4 no.4
• /
• pp.285-289
• /
• 1994

An insecticidal crystal protein gene, cryIA(c), from Bacillus thuringiensis HD-73 was integrated into the chromosome of a xanthan-producing bacterium, Xanthomonas campestris XP92. The cryIA(c) gene expression cassette was constructed that placed the gene between the trc promoter and rrnB transcriptional terminator. The $lacl^q$ gene was also included to prevent the expression of cryIA(c) gene in X campestris cells. Southem blot analysis confirmed the integration of the cryIA(c) gene expression cassette in chromosome of X campestris XP92 transconjugant. Expression of the insecticidal crystal protein was confirmed by Western blot analysis and bioassay against the larvae of Hyphantria cunea (Lepidoptera： Arctiidae) and Plutella xylostella (Lepidoptera：Plutellidae).

• Parasites, Hosts and Diseases
• /
• v.53 no.3
• /
• pp.335-339
• /
• 2015

Cryptosporidium andersoni ATP-binding cassette (CaABC) is an important membrane protein involved in substrate transport across the membrane. In this research, the nucleotide binding domain (NBD) of CaABC gene was amplified by PCR, and the eukaryotic expression vector of pEGFP-C1-CaNBD was reconstructed. Then, the recombinant plasmid of pEGFP-C1-CaNBD was transformed into the mouse intestinal epithelial cells (IECs) to study the iron transportation function of CaABC. The results indicated that NBD region of CaABC gene can significantly elevate the transport efficiency of $Ca^{2+}$, $Mg^{2+}$, $K^+$, and $HCO_3{^-}$ in IECs (P<0.05). The significance of this study is to find the ATPase inhibitors for NBD region of CaABC gene and to inhibit ATP binding and nutrient transport of CaABC transporter. Thus, C. andersoni will be killed by inhibition of nutrient uptake. This will open up a new way for treatment of cryptosporidiosis.

• Journal of Microbiology and Biotechnology
• /
• v.3 no.2
• /
• pp.65-72
• /
• 1993

A heterologous gene expression and secretion system using a methylotrophic yeast, Hansenula polymorpha was developed for the production of anticoagulant hirudin. Hirudin gene was expressed under the control of a strong and inducible methanol oxidase (MOX or AOX) promoter. The mating factor a pre-pro leader sequence of Saccharomyces cerevisiae was employed for hirudin to be secreted into the extracellular medium. Hirudin expression cassette was introduced into three strains of H. polymorpha, A16, HPBl and DLl which have different genetic backgrounds. This expression cassette was stably integrated into the host chromosomal DNA. Biologically active and mature hirudin was efficiently expressed and secreted into the extracellular medium. About 19 mg/L of hirudin was found in the culture supernatant in the case of a two-copy integrant of the strain HPBl under suboptimal culture conditions.

• Proceedings of the PSK Conference
• /
• 2003.10b
• /
• pp.160.1-160.1
• /
• 2003

In order to confirm the biological function of ORF10 in cephabacin biosynthesis gene cluster of Lysobacter lactamgenus as an ATP-binding-cassette (ABC) transporter, the gene for ORF10 was amplified and subcloned into pET-28a(+) expression vector. After gene induction with 0.5 mM IPTG at 30~! and further cultivation at $30^~$ !. for 8 hr, a lot of the recombinant ORF10 protein was produced as soluble form in cytoplasmic fraction as well as a membrane protein in the membrane fraction as likely as other ABC transporters. (omitted)

• BMB Reports
• /
• v.40 no.5
• /
• pp.656-661
• /
• 2007

In this report, we describe an optimized method for generation of ephA8 BAC transgenic mice expressing the lacZ reporter gene under ephA8 regulatory sequences. First, we constructed a targeting vector that carries a 1.2 kb ephA8 DNA upstream of its first exon, a lacZ expression cassette, a kanamycin cassette, and a 0.7 kb ephA8 DNA downstream of its first exon. Second, the targeting vector was electroporated into cells containing the ephA8 BAC and pKOBEGA, in which recombinases induce a homologous recombination between the ephA8 BAC DNA and the targeting vector. Third, the FLP plasmid expressing the Flipase was electroporated into these bacteria to eliminate a kanamycin cassette from the recombinant BAC DNA. The appropriate structures of the modified ephA8 BAC DNA were confirmed by Southern analysis. Finally, BAC transgenic mouse embryos were generated by pronuclear injection of the recombinant BAC DNA. Whole mount X-gal staining revealed that the lacZ reporter expression is restricted to the anterior region of the developing midbrain in each transgenic embryo. These results indicate that the ephA8 BAC DNA contains most, if not all, regulatory sequences to direct temporal and spatial expression of the lacZ gene in vivo.