• Korean Journal of Microbiology
• /
• v.36 no.2
• /
• pp.109-111
• /
• 2000

A 3.5 kb plasmid from Lactobacillus. casei ssp. cosei NCIB 4114 was isolated and E. cali-L. casei shuttle vectors were constructed containing this plasmid. Transformation by electroporation was successful with all the plasmids constructed. Optimized condition for the electroporation was established with efficiency level of $2{\times}10^5$ transformants per $\mu$g of vector DNA. Successful introduction of those shuttle vectors enable to these vectors as food grade vector for lactic acid bacteria.

• Korean Journal of Microbiology
• /
• v.29 no.3
• /
• pp.149-154
• /
• 1991

Escherichia coli/Corynebacterium glutamicum shuttle vectors, pECCG1 and pECCG2 were constructed by joining a 3.00 kb cryptic plasmid pCB 1 from C. glutamicum and a 3.94 kb plasmid pACYC 177 from E. coli. By trimming unessential parts and introducing mulitiple cloning site into the plasmid pECCG 1, a plasmid pECCG122(5.1kb) was constructed. All the shuttle vectors were stably maintained in C. glutamicum up to about 40 generations irrespective of kanamycin addition in the medium. Threonine operon (homoserine dehydrogenase/homoserine kinase) and dapA gene (dihydrodipicolinate synthetase) of C. glutamicum were cloned into the plasmid pECCG122, and the resultant plasmids were designated pTN31 and pDHDP19, respectively. They were used to study the effect of cloned foreign gene on the stability of the plasmid pECCG122. Plasmids pTN31 and pDHDP19 were segregated rapidly from C. glutamicum when cultured in the medium without kanamycin. In medium with $50\mu${\g/ml} of kanamycin, their segregation rates were much slower than those in medium without kanamycin, but the danamycin addition didn't guarantee the complete maintenance of the plasmids in C. glutamicum.

• Korean Journal of Microbiology
• /
• v.25 no.4
• /
• pp.262-273
• /
• 1987

The replication region of the chloramphenical resistance plasmid pSBK203 of Staphylococcus aureus was cloned using pBR322 and pBD9 as vectors. Cloned replication tegion and chloramphenicol resistance gene were recombined to pBR322. The reconstructed vector behaved as a shuttle vector for E. coli and B. subtilis.

• Microbiology and Biotechnology Letters
• /
• v.15 no.5
• /
• pp.319-327
• /
• 1987

In order to develop useful plasmid vectors for Zymomonas cells, attempts were made to isolate natural plasmids from Z. mobilis ATCC10988. Among a few plasmids isolated, a small plasmid of 3.9 Kb size was chosen and designated as pZM3. By introducing the replication origin of pZM3 into pBR325, a hybrid plasmid vector of 8.4 Kb size, pHZ22, was constructed. This vector contained chloramphenicol resistant gene as a selectable marker and proved to be conjugally transmissible and stably maintained in Z. mobilis. Tetracycline resistant gene was isolated from RP4 and introduced into pHZ22 to make a new vector called pHZT224 of 10.7 Kb size. Through n series of experiments, it was evident that these plasmid vectors containing selectable markers of chloramphenicol and tetracycline resistance were shuttle vectors functional in Z. mobilis as well as E. coli.

• Microbiology and Biotechnology Letters
• /
• v.29 no.1
• /
• pp.1-11
• /
• 2001

Lactic acid bacteria (LAB) are widely used for various food fermentation. With the recent advances in modern biotechnology, a variety of bio-products with the high economic values have been produced using microorganisms. For molecular cloning and expression studies on the gene of interest, E. coli has been widely used mainly because vector systems are fully developed. Most plasmid vectors currently used for E, coli carry antibiotic-resistant markers. As it is generally believed that the antibiotic resistance markers are potentially transferred to other bacteria, application of the plasmid vectors carrying antibiotic resistance genes as selection markers should be avoided, especially for human consump-tion. By contrast, as LAB have some desirable traits such that the they are GRAS(generally recognized as safe), able to secrete gene products out of cell, and their low protease activities, they are regarded as an ideal organism for the genetic manipulation, including cloning and expression of homologous and heterologous genes. However, the vec-tor systems established for LAB are stil insufficient to over-produce gene products, stably, limiting the use of these organisms for industrial applications. For a past decade, the two popular plasmid vectors, pAM$\beta$1 of Streptococcus faecalis and pGK12 theB. subtilis-E. coli shuttle vector derived from pWV01 of Lactococcus lactis ssp. cremoris wg 2, were most widely used to construct efficient chimeric vectors to be stably maintained in many industrial strains of LAB. Currently, non-antibiotic markers such as nisin resistance($Nis^{r}$ ) are explored for selecting recombi-nant clone. In addition, a gene encoding S-layer protein, slp/A, on bacterial cell wall was successfully recombined with the proper LAB vectors LAB vectors for excretion of the heterologous gene product from LAB Many food-grade host vec-tor systems were successfully developed, which allowed stable integration of multiple plasmid copies in the vec-mosome of LAB. More recently, an integration vector system based on the site-specific integration apparatus of temperate lactococcal bacteriophage, containing the integrase gene(int) and phage attachment site(attP), was pub-lished. In conclusion, when various vector system, which are maintain stably and expressed strongly in LAB, are developed, lost of such food products as enzymes, pharmaceuticals, bioactive food ingredients for human consump-tion would be produced at a full scale in LAB.

• The Korean Journal of Pesticide Science
• /
• v.2 no.1
• /
• pp.91-96
• /
• 1998

For the expression of the cry11Aa gene highly toxic to dipteran insects, we constructed two cyanobacteria-Escherichia coli(E. coli) shuttle vectors, pCYASK5-l and pCYASK5-2. These vectors were transformed into E. coli and selected with kanamycin. The expression of the cry11Aa gene in E. coli was characterized by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Two E. coli transformants harboring pCYASK5-1 and pCYASK5-2 expressed the cry11Aa gene in size of 72 kDa and 64 kDa, respectively and showed over 89% mortality against Culex pipiens larvae.

• Journal of Microbiology and Biotechnology
• /
• v.1 no.1
• /
• pp.37-44
• /
• 1991

A shuttle promoter-probe vector, pEB203, was derived from pBR322, pPL703 and pUB110. Using the vector, a useful DNA fragment, 319 bp EcoRI fragment, having strong promoter activity has been cloned from Bacillus subtills chromosomal DNA. Selection was based on chloramphenicol resistance which is dependent upon the introduction of DNA fragments allowing expression of a chloramphenicol acetyl transferase gene. The nucleotide sequence of the 319 bp fragment has been determined and the putative -35 and -10 region, ribosome binding site, and ATG initiation codon were observed. This promoter was named EB promoter and the resultant plasmid which can be used as an expression vector was named pEBP313. The crystal protein gene from B. thuringiensis subsp. kurstaki HD-73 was cloned downstream from the EB promoter without its own promoter. When the resultant plasmid, pBT313, was introduced into Escherichia coli and B. subtilis, efficient synthesis of crystal protein was observed in both cells, and the cp gene expression in B. subtilis begins early in the vegetative phase. The cell extracts from both clones were toxic to Hyphantria cunea larvae.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.6
• /
• pp.850-855
• /
• 2013

Finding new antimicrobial activities by functional metagenomics has been shown to depend on the heterologous host used to express the foreign DNA. Therefore, efforts are devoted to developing new tools for constructing metagenomic libraries in shuttle vectors replicatable in phylogenetically distinct hosts. Here we evaluated the use of the Escherichia coli-Bacillus subtilis shuttle vector pHT01 to construct a forest-soil metagenomic library. This library was screened in both hosts for antimicrobial activities against four opportunistic bacteria: Proteus vulgaris, Bacillus cereus, Staphylococcus epidermidis, and Micrococcus luteus. A new antibacterial activity against B. cereus was found upon screening in B. subtilis. The new antimicrobial agent, sensitive to proteinase K, was not active when the corresponding DNA fragment was expressed in E. coli. Our results validate the use of pHT01 as a shuttle vector and B. subtilis as a host to isolate new activities by functional metagenomics.

• Korean Journal of Microbiology
• /
• v.30 no.3
• /
• pp.218-224
• /
• 1992

Recombinant plasmid shuttle vectors were constructed for genetic studies on the oxidation of carbon monoxide by carboxydobacteria. Two vectors. pYK322 (7.2 kb, Ap'. Tc') and pYK 324 (7.2 kb, Ap', Tc'), were constructed using pBR322 and pYK100. a small plasmid in Pseudomonas carbo,xydovorans. Four plasmids. pYK2IO (5.2 kb. Cm'), pYK220 (5.2 kb, Cmr), pYK230 (5.2 kb, Cm'), and pYK232 (5.2 kb. Cm'), were constructed using pACYC184 and pYK100. Transformation of several carboxydobacteria with pYK322 and pYK220 was round to be efficient when the cells were transformed by the methoti of Bagdasarian and Timmis (Curr. Top. Microbiol. Immunol. 96:47-67. 1982) with several modifications; cells growing on 0.2% succinate were harvested at the mid-exponential phase. 10 mM RbCl in transformation solution was substituted with 100 mM KCI. cclls in transformation solution were incubated for 12 h at 4'C before addition of DNA and heat shock was carried out for 3 min at 45$^{\circ}$C. Plasmid vectors used for transformation, however. were not detected from antibiotics-resistant transformants, suggesting that the vectors may be integrated into the chromosomal DNA.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.11 no.2
• /
• pp.125-127
• /
• 2005

In order to improve the transformation efficiency of the Bacillus thuringiensis (Bt)-Escherichia coli (E. coli) shuttle vector, pHT3101, we intended to minimize replication origin of Bt in pHT3101. For this, two modified shuttle vectors, pHT1K and pHT261, in which 2.9 kb of replication origin of Bt were shortened to 1 kb and 261 bp, respectively as previously reported. Whereas the pHT1K could efficiently transform Bt into the antibiotic resistant, no transformants were obtained with pHT261. Furthermore, pHT1K showed higher transformation efficiency compared to that of parent vector, pHT3101. Therefore, pHT1K might be a very useful Bt-E. coli shuttle vector carrying minimal replication origin of Bt.