BCG has been one of the vehicles for multi-recombinant vaccine. However, low transformation efficiency of BCG with plasmid DNA hampered studies involving expression of foreign antigens in BCG. In an effort to determine the optimal conditions, this study was initiated to investigate factors involved in the transformation of BCG with a Mycobacterium-Escherichia coli shuttle vector, pYUB18, by electroporation. Mycobacterium bovis BCG (strain 1173P2) was grown in Middlebrook (M) 7H9 broth containing albumin-dextrose-catalase and 0.05% tween 80, and transformed BCG was grown in M7H10 agar containing kanamycin for counting viable cells. Pretreatment of BCG with 10 mM CaCl2 improved the transformation efficiency, but overnight incubation of BCG with 1% glycine did not. The transformation efficiency in BCG also varied depending on voltage, resistance, and DNA concentration. The maximum transformation efficiency was obtained when the infinity resistance, 12.5 Kv/cm, and 100 ng of DNA were used, and reached 1.4 x 10(5) CFU/microgram of plasmid DNA, which is about 3-100 times greater than those from previous reports. The transformation conditions described in this study, therefore, will give us a better position for employing BCG as a vehicle for developing multi-recombinant vaccines.
Calcium Chloride/pharmacology ; Comparative Study ; DNA/metabolism ; Electrophysiology ; Electroporation* ; Escherichia coli/genetics* ; Genetic Vectors* ; Glycine/pharmacology ; Mycobacterium/genetics* ; Mycobacterium bovis/genetics* ; Osmolar Concentration ; Transformation, Bacterial/physiology* ; Transformation, Bacterial/drug effects

The objective of this study was to characterize the oxygen dependent regulation of pyruvate oxidase (SpxB) gene expression and protein production in Streptococcus sanguinis (S. sanguinis). SpxB is responsible for the generation of growth-inhibiting amounts of hydrogen peroxide (H2O2) able to antagonize cariogenic Streptococcus mutans (S. mutans). Furthermore, the ecological consequence of H2O2 production was investigated in its self-inhibiting ability towards the producing strain. Expression of spxB was determined with quantitative Real-Time RT-PCR and a fluorescent expression reporter strain. Protein abundance was investigated with FLAG epitope engineered in frame on the C-terminal end of SpxB. Self inhibition was tested with an antagonism plate assay. The expression and protein abundance decreased in cells grown under anaerobic conditions. S. sanguinis was resistant against its own produced H2O2, while cariogenic S. mutans was inhibited in its growth. The results suggest that S. sanguinis produces H2O2 as antimicrobial substance to inhibit susceptible niche competing species like S. mutans during initial biofilm formation, when oxygen availability allows for spxB expression and Spx production.
Antibiosis ; physiology ; Bacterial Proteins ; biosynthesis ; genetics ; Epitopes ; genetics ; Gene Expression Regulation, Bacterial ; Hydrogen Peroxide ; metabolism ; pharmacology ; Oligopeptides ; Oxygen ; metabolism ; Peptides ; genetics ; Pyruvate Oxidase ; biosynthesis ; genetics ; Streptococcus mutans ; drug effects ; Streptococcus sanguis ; enzymology ; genetics ; growth & development ; Transformation, Bacterial

Obtaining marker-free plants with high efficiency will benefit the environmental release of transgenic crops. To achieve this point, a binary vector pNB35SVIP1 with three T-DNAs was constructed by using several mediate plasmids, in which one copy of bar gene expression cassette and two copies of VIP1 gene expression cassette were included. EHA101 Agrobacterium strain harboring the final construct was applied to transform soybean (Glycine max) cotyledon nodes. Through 2 - 3 months regeneration and selection on 3 - 5mg/L glufosinate containing medium, transgenic soybean plants were confirmed to be obtained at 0.83% - 3.16%, and co-transformation efficiency of both gene in the same individual reached up to 86.4%, based on southern blot test. By the analysis of PCR, southern blot and northern blot combining with leaf painting of herbicide in T1 progenies, 41 plants were confirmed to be eliminated of bar gene with the frequency of 7.6% . Among the T1 populations tested, the loss of the alien genes happened in 22.7% lines, the silence of bar gene took place in 27.3% lines, and VIP1 gene silence existed in 37.1% marker-free plants. The result also suggested that the plasmid with three T-DNAs might be an ideal vector to generate maker-free genetic modified organism.
Aminobutyrates ; pharmacology ; Blotting, Northern ; Blotting, Southern ; Cotyledon ; drug effects ; genetics ; physiology ; DNA, Bacterial ; genetics ; Gene Expression Regulation, Plant ; Genes, Plant ; genetics ; Genetic Vectors ; genetics ; Herbicide Resistance ; genetics ; Herbicides ; pharmacology ; Plant Leaves ; drug effects ; genetics ; physiology ; Plants, Genetically Modified ; drug effects ; genetics ; physiology ; Regeneration ; drug effects ; genetics ; physiology ; Rhizobium ; genetics ; Soybeans ; drug effects ; genetics ; physiology ; Transformation, Genetic

The rice blast fungus Magnaporthe grisea causes one of the most destructive diseases of rice around the world. Significant progresses have been made recently in genomics studies of the fungus, opening new era of the functional genomics which requires to generate a large scale of gene knockout mutants. It has been demonstrated that T-DNA insertional mutagenesis is a powerful tool of functional genomics not only for plants but also for fungi. In this paper, we optimized the conditions for T-DNA insertional mutagenesis of M. grisea using Agrobacterium tumefaciens-mediated transformation (ATMT) approach. We employed the binary vector pBHtl constructed by Dr. S. Kang's laboratory at the Pennsylvania State University, which carries the bacterial hygromycin B phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker to transform the conidia of M. grisea. We optimized the conditions for T-DNA insertional mutagenesis including the medium, dosage of hygromycin B, cefotaxime and carbenicillin to select the transformants and inhibit the growth of A. tumefaciens after co-culturing. The dosage to inhibit non-transformants could vary from 200-600microg/mL among different M. grisea isolates so that the optimal dosage of the antibiotics should be decided according to isolates. Rice polished agar medium was found the best selection medium which would facilitate the mutant sporulation and minimize the contamination chance. In average, about 500 transformants could be obtained when transforming 1 x 10(6) spores at the optimum condition, among which 85% had T-DNA insertion detected by polymerase chain reaction (PCR) and thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). Fifteen out of 1520 transformants showed mutation in colony morphology. Within 58 randomly selected mutants, it was found that there were 4 sporulation-decreased mutants, 8 less germination mutants and 9 appressorium defective mutants. Several virulent mutants to C101LAC(Pi-1)and 75-1-127(Pi-9)were also obtained which would facilitate cloning the corresponding avirulence genes.
Agrobacterium tumefaciens ; genetics ; Bacterial Proteins ; genetics ; Carbenicillin ; pharmacology ; Cefotaxime ; pharmacology ; DNA, Bacterial ; genetics ; physiology ; Genetic Vectors ; genetics ; Hygromycin B ; pharmacology ; Magnaporthe ; drug effects ; genetics ; Mutagenesis, Insertional ; methods ; Mutation ; Oryza ; microbiology ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics ; Transformation, Genetic ; genetics ; physiology

OBJECTIVETo transform the antimicrobial peptide fusion gene of cecropin B and rabbit NP-1(CN) into Houttuynia cordata to improve its antimicrobic capability.

METHODThe fusion gene of CN designed and synthesized artificially was recombined with expression vector pBI121. The recombined vector was transformed to Agrobacterium tumefaciens LBA4404, by which CN gene was transformed to the explants of H. cordata. The transgenic regeneration plantlets were selected by kanamycin and rapid screening PCR. The transgenic plants were identified by PCR-Southern of genomic DNA and RT-PCR. The disease resistances were detected by antibacterial zone trail of leaf extracts to E. coli K12 and infection by Rhizoctonia solani.

RESULTGene of interesting CN was inserted into genomic DNA and expressed in transformed H, cordata, whose resistance to E. coli K12 and Rh. solani was stronger than that of the non-transformed control.

CONCLUSIONThe fusion gene CN can improve antimicrobic capability of transformed H. cordata.

Animals ; Anti-Bacterial Agents ; immunology ; pharmacology ; C-Reactive Protein ; genetics ; metabolism ; pharmacology ; Houttuynia ; genetics ; immunology ; microbiology ; Immunity, Innate ; Insect Proteins ; genetics ; immunology ; pharmacology ; Nerve Tissue Proteins ; genetics ; metabolism ; pharmacology ; Plant Diseases ; immunology ; microbiology ; Plants, Genetically Modified ; genetics ; immunology ; microbiology ; Rabbits ; Recombinant Fusion Proteins ; genetics ; immunology ; pharmacology ; Rhizoctonia ; physiology ; Transformation, Genetic