Sepiolite--an inexpensive, resourceful, fibrous yet inoffensive mineral--made DNA transformation rapid, simple and efficient but the mechanism for DNA transformation was still unclear. Through RNA competition test, we proposed the different transforming mechanisms from the previous report. Meanwhile, we optimized the transforming method and could transfer a colony stored at 4 degrees C for a month with plasmid through sepiolite fibers. The cells could be transformed well without competent cells preparation or incubation process. In sum, this was a novel potential transforming method, which could be explored further if the chemical method and electroporation could not be used.
DNA, Bacterial ; chemistry ; genetics ; Electroporation ; methods ; Magnesium Silicates ; chemistry ; Mineral Fibers ; Nanofibers ; chemistry ; Transformation, Bacterial

Bacterial Proteins ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; physiology ; Glutathione ; metabolism ; Listeria monocytogenes ; chemistry ; genetics ; metabolism ; Peptide Termination Factors ; chemistry ; genetics ; metabolism

In order to optimize the conditions of construction BAC library, the transformation efficiency of E. coli DH10B was studied in this paper. Our data prove much higher competence of electroporation (reaches 2.19 x 10(10) cfu/microg pUC19 DNA) when harvesting the cells between an OD550 of 0.7 - 0.8. Five different electric field strength (from 9 kV/cm to 25 kV/cm) and three different sized plasmid vector DNAs including pUC19 DNA, pECBAC1 DNA and pCLD04541 DNA, as well as three bacterial artificial chromosomes (BACs) ranging from 40 to 190 kb and their mixture were used to discover the transformation efficiency changes under various conditions. Our data show maximum transformation efficiency and optimal electric field strength of plasmid DNAs drop dramatically with increasing size of the DNA. Molecules of 190 kb transform more than 50-fold less well, on a molar basis, than molecules of 40 kb. And the optimal voltage gradient is strongly dependent on the different sized molecules, for instance, pUC19 reaches the highest transformation efficiency at 21 kV/cm, while the 180 kb BAC DNA gets its best efficiency at 13 kV/cm. This paper demonstrates that conditions may be selected which increase the average size of BAC clones generated by electroporation and could be widely applied in large-insert genome library construction.
Chromosomes, Artificial, Bacterial ; genetics ; DNA, Bacterial ; chemistry ; genetics ; Electroporation ; methods ; Escherichia coli ; genetics ; Molecular Weight ; Plasmids ; genetics ; Transformation, Genetic

DNA stable-isotope probing (DNA-SIP) is a recently developed method with which the incorporation of stable isotope from a labeled substrate is used to identify the function of microorganisms in the environment. The technique has now been used in conjunction with metagenomics to establish links between microbial identity and particular metabolic functions. The combination of DNA-SIP and metagenomics not only permits the detection of rare low-abundance species from metagenomic libraries but also facilitates the detection of novel enzymes and bioactive compounds. We summarize recent progress in SIP-metagenomic techniques and applications and discuss prospects for this combined approach in environmental microbiology and biotechnology.
Animals ; DNA ; genetics ; DNA Probes ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; genetics ; metabolism ; Humans ; Isotope Labeling ; methods ; Metagenomics ; methods ; Molecular Probe Techniques ; Sequence Analysis, DNA ; methods

As the dominant antigens, early secreted antigenic target 6 (ESAT6, E6) and culture filtrate protein 10 (CFP10, C10) had once been the focus of tuberculosis (TB) vaccine due to their capability of inducing strong cell immune response in the host. They are also endowed with promising future of prevention against and diagnosis of TB. In this review, we systematically introduce recent research progress of E6 and C10, especially in structure-function, biological characteristics, protein expression and secretion, host immunity and vaccine development, and the prospects of their application are also discussed.
Antigens, Bacterial ; chemistry ; genetics ; immunology ; Bacterial Proteins ; chemistry ; genetics ; immunology ; Humans ; Immunodominant Epitopes ; immunology ; Molecular Biology ; Peptide Fragments ; chemistry ; genetics ; immunology ; Tuberculosis Vaccines ; genetics ; immunology ; Vaccines, DNA ; immunology

In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth (Luria-Bertani agar plates for 12-18 h) and lysis conditions (4 h incubation with 500 µg/mL lysozyme) produced sharp bands on the gel. Restriction enzyme NotI was chosen as the most suitable. Twenty-two isolates were analyzed by NotI digestion, using three electrophoretic parameters (EPs). The EP-a was optimal for distinguishing between isolates. The optimized protocol could be completed within 40 h which is a significant improvement over the previous methods.
Bacillus cereus ; genetics ; isolation & purification ; Bacteriological Techniques ; DNA, Bacterial ; chemistry ; genetics ; Electrophoresis, Gel, Pulsed-Field ; methods

OBJECTIVETo establish a new nucleic acid hybridization detection technique which may be used in medical genechips.

METHODSThe specific DNA fragment was detected by sequential two hybridization of fluorescence probe with template DNA and fixed DNA probe.

RESULTSFluorescence probe two-hybridization (FPTH) was applied to genechips for the detection of sex-transmitted pathogens from culture strains, and the results showed that the values of fluorescence density of the positive groups decreased remarkably when compared with those of the negative group. Both the sensitivity and specificity for detecting clinical samples are higher than 90%. There is no need of any additional reagent in hybridization procedure, and the hybridization detection can be accomplished in 40 minutes.

CONCLUSIONThe FPTH technique is rapid, simple and reliable, it can also make the clinical detection process completely automatic and integrative.

DNA Probes ; chemistry ; genetics ; DNA, Bacterial ; genetics ; Fluorescent Dyes ; chemistry ; Humans ; Neisseria gonorrhoeae ; genetics ; Nucleic Acid Hybridization ; methods ; Ureaplasma urealyticum ; genetics

Following Escherichia coli lysis with alkali, cetyltrimethylammonium bromide (CTAB) was directly titrated into the supernatant. An easy and feasible technology for plasmid purification was established with the optimized proportion between the quantity of CTAB and plasmid, combined with the specific solution for DNA release and TritonX-114 for endotoxin removal. Quality detection showed that the purified plasmid was free of contamination of host RNA. The host bacterial genomic DNA, endotoxin and bacterial protein were less than 10 microg, 50 EU and 10 microg per mg plasmids, respectively. The ratio of OD260/OD280 was between 1.75-1.85. Eighty percent of the prepared plasmids were presented in the supercoiled form. The plasmid purified with this technology can satisfy all criteria stipulated by FDA. The main advantages of the technology include the avoidance of animal-derived enzymes such as ribonucleases A, Proteinase K and toxic reagents like chloroform and phenol. In addition, the technology has low cost and no pollution.
Cetrimonium Compounds ; chemistry ; Chemical Precipitation ; DNA ; genetics ; isolation & purification ; DNA, Bacterial ; genetics ; isolation & purification ; Escherichia coli ; chemistry ; genetics ; Plasmids ; genetics ; isolation & purification

OBJECTIVEIn order to find out the current situation of tick-borne spotted fever in the area of Changbai mountain, Jilin province.

METHODSIn this study, a polymerase chain reaction (PCR) method was developed with primers R. rOmpA 190.70p and R. rOmpA 190.701n designed on the basis of rOmpA gene, which is specific for examining spotted fever group Rickttsiaes (SFGR). Six hundred nighty-three ticks were tested and a positive PCR product amplified from D. silvarum specimen (named JL-02) was cloned and sequenced.

RESULTSThe SFGR DNA was detected from D. silvarum, Haemaphysalis concinna with the positive rates were 53.81% and 7.41% respectively. Its nucleotide sequence of 587 bp rOmpA and derived amino-acids showed 100.00% similarity with nucleotide sequence of DnS 14 and 99.00% with DnS 28 from the Former Soviet Union according to the result of BLUST and CLUSTAL, which was differential from the DNA sequences of strains previously detected in China.

CONCLUSIONThe natural focus of tick-borne spotted fever did exist in the area of Changbai mountain. The DNA sequence of SFGR was similar to that of DnS 14, which was first reported in China.

Bacterial Outer Membrane Proteins ; genetics ; China ; DNA, Bacterial ; chemistry ; genetics ; Humans ; Phylogeny ; Polymerase Chain Reaction ; Rickettsia Infections ; microbiology ; Rickettsieae ; classification ; genetics ; Sequence Analysis, DNA

Degenerate PCR primers were designed by multiple alignment of the protein sequences of known structural genes encoding the catalytic subunits of NiFe-hydrogenases obtained from Swiss-Prot Protein Sequence Database through CLUSTAL-W software and compared for conserved sequence motifs. An amplified PCR product 1 kb in size was obtained from the genomic DNA of Klebsiella pneumoniae using a set of degenerate primers, and then inverse PCR technique was used to obtain the full hydrogenase coding region. A predicted secondary structure and 3D structural model were constructed by homology modeling and docking. On the basis of these results, it was inferred that NiFe-hydrogenase from Klebsiella pneumoniae belongs to the membrane-bound H2 evolving hydrogenase group (Ech hydrogenase group).
Bacterial Proteins ; chemistry ; genetics ; Cloning, Molecular ; Codon ; genetics ; DNA, Bacterial ; chemistry ; genetics ; Databases, Protein ; Hydrogen Bonding ; Hydrogenase ; chemistry ; genetics ; Klebsiella pneumoniae ; enzymology ; genetics ; Models, Molecular ; Molecular Sequence Data ; Protein Conformation ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Sequence Analysis, DNA