Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.
Chromosomes, Bacterial ; genetics ; DNA ; Endonucleases ; metabolism ; Escherichia coli ; genetics ; Genetic Engineering ; methods ; Recombination, Genetic ; Sequence Deletion

OBJECTIVETuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis (M. tuberculosis) multilocus sequence analysis (MLSA) was established for the phylogenetic and epidemiology analysis.

METHODSTo establish the scheme of M. tuberculosis MLSA, the genome of H37Rv, CCDC5079 and CCDC5180 were compared, and some variable genes were chosen to be the MLSA typing scheme. 44 M. tuberculosis clinical isolates were typed by MLSA, IS6110-RFLP, and soligotyping, to evaluate the MLSA methods.

RESULTSAfter comparison of the genome, seven high discrimination gene loci (recX, rpsL, rmlC, rpmG1, mprA, gcvH, ideR) were chosen to be the MLSA typing scheme finally. 11 variable SNP sites of those seven genes were found among the 44 M. tuberculosis isolate strains and 11 sequence types (STs) were identified. Based on the Hunter-Gaston Index (HGI), MLSA typing was not as good for discrimination at the strain level as IS6110-RFLP, but the HGI was much better than that of spoligotyping. In addition, the MEGA analysis result of MLSA data was similar to spoligotyping/PGG lineage, showing a strong phylogenetic signal in the modern strains of M. tuberculosis. The MLSA data analysis by eBURST revealed that 4 sequence types (ST) came into a main cluster, showing the major clonal complexes in those 44 strains.

CONCLUSIONMLSA genotyping not only can be used for molecular typing, but also is an ideal method for the phylogenetic analysis for M. tuberculosis.

Chromosome Mapping ; Chromosomes, Bacterial ; Gene Expression Regulation, Bacterial ; Genome, Bacterial ; Genotype ; Multilocus Sequence Typing ; methods ; Mycobacterium tuberculosis ; genetics ; metabolism

To construct a vector for DNA vaccine and protein expression by using chromosome-plasmid balanced lethal system which was based on the thyA+ gene/deltathyA Escherichia coli. The thyA genes from Escherichia coli and Vibrio cholerae were amplified by polymerase chain reaction and cloned into pCDNA3 by replacing ampilicilin resistant gene. Multiple cloning sites, the prokaryotic replicon, CMV promoter and the boving growth hormone polyA signal were also included in the vectors. Two new non-antibiotic recombinant plasmids renamed as pcDNATE and pcDNATC which had the nutritional marker as thyA were constructed and were transformed respectively into the deltathyA derivative of E. coli K-12 strain DY330-TI, then two chromosome-plasmid balanced systems for E. coli based on the thyA were developed. To test the efficiency and stability of the newly constructed chromosome-plasmid balanced lethal system, a reporter gene--red fluorescent protein (DsRed2) gene was cloned into pcDNATE, pcDNATC and expressed as fusion to the c-myc. The two recombinant plasmids, pcDNATE-DsRed2, pcDNATC-DsRed2, were transfected into HEK293 solely and DsRed2-myc was detected by the fluorescence microscope assay and western-blot. Meanwhile, the loss of recombinant plasmids were not seen in cultures without thymidine after 20 generations. The chromosomal-plasmid balanced lethal system is proved to be an effective vector system for the expression of target genes and share the same stability with the antibiotic-resistant plasmid vector system. It holds great potential in gene vaccine vector because obviating the weakpoints of the drug resistance marker during application.
Bacterial Proteins ; genetics ; metabolism ; Blotting, Western ; Cell Line ; Chromosomes, Bacterial ; genetics ; Escherichia coli ; enzymology ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Microscopy, Fluorescence ; Models, Genetic ; Plasmids ; genetics ; Polymerase Chain Reaction ; Thymidylate Synthase ; genetics ; metabolism ; Vibrio cholerae ; enzymology ; genetics ; metabolism

To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.
Bacteriophage lambda ; genetics ; Chromosomes, Bacterial ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Knock-In Techniques ; methods ; Genes, Reporter ; genetics ; Lac Operon ; genetics ; Luciferases ; genetics ; Recombination, Genetic ; genetics

Genetic engineering on macrolide antibiotics was a new field in recent years and more than 100 novel polyketides had been produced until then. Using genomic DNA of S. erythraea A226 as a template, about 3.2 kb DNA fragment without KR6 domain was amplified by overlapping PCR technique and cloned into pWHM3 carrier, which resulted in the construction of homologous recombinant plasmid pWHM2201. Plasmid pWHM2201 was introduced into protoplasts of S. erythraea A226 by PEG-mediated transformation and integrated into the gene locus for erythromycin biosynthesis. After integrants grew for two generations on R3M media without Tsr, they were protoplasted and grown on R3M plates. By PCR identification, 8 mutants without KR6 domain were selected out and named S. erythraea M(1-8). With the identification of mass spectrometry, it was proved that S. erythraea M1 synthesized a novel ketolide compound 3-deoxy-3-oxo-erythronolide B.
Anti-Bacterial Agents ; biosynthesis ; chemistry ; Chromosomes, Bacterial ; Erythromycin ; analogs & derivatives ; biosynthesis ; chemistry ; Genetic Engineering ; Ketolides ; Molecular Structure ; Multienzyme Complexes ; genetics ; Saccharopolyspora ; enzymology ; genetics ; metabolism

OBJECTIVETo establish chromosome conformation capture (3C) strategy and to use this method for exploring the effect of chromosome conformation on human alpha-globin gene expression in the human alpha-globin transgenic mouse.

METHODSHomozygous human alpha-globin transgenic male mouse was crossed with KM female mouse. The 14.5-day post-coitum (dpc) embryos were used for the isolation of fetal liver and fetal brain cells. Homogeneous single-cell suspension was treated with formaldehyde to crosslink the chromatin conformation in the nuclear. The cross-linked chromatin compound was digested with Nco I and then ligated with T4 DNA ligase. The ligated compound was reversely cross-linked and then the ligated genomic DNA was purified for PCR analysis. The primers were designed along the two sides of cut and ligated sites. Semi-quantitative PCR was used to analyze the chromosome conformation of the whole human alpha-globin gene locus in fetal liver and fetal brain cells.

RESULTSWhen HS40 fragment was used as the fixed fragment, in fetal brain cells, the ligation frequencies of HS40 fragment with other fragments were decreased as the linear distances to HS40 fragment were increasing; while in fetal liver cells, two active genes (alpha1 and alpha2) fragments showed higher ligation frequencies with HS40 fragment than other fragments. However, the fragment containing an inactive gene (xi) displayed the comparable low ligation frequency as that in fetal brain. When alpha2 fragment was used as the fixed fragment, similarly, in fetal brain cells the ligation frequencies of alpha2 fragment with other ones were decreased as the linear distances increasing; when in fetal liver cells, it showed higher ligation frequencies with two upstream regulatory elements (HS 40 and 33). However, it showed a little bit lower ligation frequency with another two upstream regulatory elements (HS10 and 8) than those in fetal brain.

CONCLUSIONIn fetal liver cells, the distant regulatory elements are in close proximity to the downstream of the expressed globin genes through looping out, the interval region; however, in fetal brain, they were not in vicinity to the expressed globin genes.

Animals ; Brain ; metabolism ; Chromosomes, Artificial, Bacterial ; Chromosomes, Mammalian ; chemistry ; Female ; Gene Expression Regulation ; Humans ; Liver ; metabolism ; Male ; Mice ; Mice, Transgenic ; Nucleic Acid Conformation ; Regulatory Sequences, Nucleic Acid ; alpha-Globins ; biosynthesis ; genetics

The aim of this study was to construct the complete genome of Marek's disease virus serotype 814 strain as an infectious bacterial artificial chromosome (BAC). Using self-designed selection marker Eco-gpt (1.3 kb) and BAC vector pBeloBAC11 (7.5 kb), we constructed the transfer plasmid pUAB-gpt-BAC11. The plasmid pUAB-gpt-BAC11 and MDV total-DNA were cotransfected into secondary CEFs; we put the virus-containing cells in selection medium for eight rounds and obtained purified recombinant viruses. Recombinant viral genomes were extracted and electroporated into E. coli, BAC clones were identified by restriction enzyme digestion and PCR analysis. Finally, we obtained 38 BAC clones, DNA from various MDV-1 BACs was transfected into CEFs, and recombinant virus was reconstituted by transfection of MDV-BAC2 DNA. We successfully cloned the complete genome of MDV-1814 strain as an infectious bacterial artificial chromosome. With these cloned genomes, a revolutionary MDV-DNA engineering platform utilizing RED/ET recombination system was constructed successfully, which can help the understanding of MDV gene functions and promote the using of MDV as a vector for expressing foreign genes. In addition, it opens the possibility to generate novel MDV-1 vaccines based on the BACs.
Animals ; Chickens ; immunology ; virology ; Chromosomes, Artificial, Bacterial ; genetics ; Cloning, Molecular ; DNA, Recombinant ; genetics ; DNA, Viral ; genetics ; Fibroblasts ; metabolism ; Genetic Engineering ; methods ; Mardivirus ; classification ; genetics ; physiology ; Serotyping ; Transfection ; Viral Proteins ; genetics ; physiology ; Virus Replication

Genetic manipulation of human pluripotent stem cells (hPSCs) provides a powerful tool for modeling diseases and developing future medicine. Recently a number of independent genome-editing techniques were developed, including plasmid, bacterial artificial chromosome, adeno-associated virus vector, zinc finger nuclease, transcription activator-like effecter nuclease, and helper-dependent adenoviral vector. Gene editing has been successfully employed in different aspects of stem cell research such as gene correction, mutation knock-in, and establishment of reporter cell lines (Raya et al., 2009; Howden et al., 2011; Li et al., 2011; Liu et al., 2011b; Papapetrou et al., 2011; Sebastiano et al., 2011; Soldner et al., 2011; Zou et al., 2011a). These techniques combined with the utility of hPSCs will significantly influence the area of regenerative medicine.
Cell Line ; Chromosomes, Artificial, Bacterial ; genetics ; Deoxyribonucleases ; genetics ; Dependovirus ; genetics ; Gene Targeting ; methods ; Genetic Engineering ; methods ; Genetic Vectors ; Genome, Human ; Humans ; Mutagenesis, Insertional ; Mutation ; Plasmids ; Pluripotent Stem Cells ; cytology ; metabolism ; Zinc Fingers ; genetics

Based on the sequence of BAC (Bacterial Artificial Chromosome) along with the Cre/lox P system, the gene-targeting vectors to multiple loci of the repetitive internal transcribed spacers between rDNA genes in Leghorn chicken were constructed. The key material of multiple loci gene targeting in vivo would be obtained. First, the plasmid of pYLSV-TDN with TK, HRDS2, and Neo genes was constructed. The TK-HRDS2-Neo DNA fragment obtained from the plasmid of pYLSV-TDN was digested by Not I/HindIII and inserted into the upstream of the lox P site of BAC plasmid for obtaining the selective vector of BAC-TDN. The expression vector of pYLVS-GID with EGFP, hIFN genes, and HRDS1 was then obtained. The plasmid of BAC-TDN-VS-GID was obtained by cotransformation of the selective vector of BAC-TDN and the expression vector of pYLVS-GID to E. coli NS3529 through the action of Cre/lox P system. The gene-targeting vector of BAC-TDN-GID to multiple loci of the ITS region in Leghorn chicken was obtained by cleaving the sequence of pYLVS with the homing endonuclease of I -Sce I and ligating with the linker of LS. The insertion and the insert direction of DNA fragments were identified by restriction digestion or PCR and sequencing in each clone. The significance of the technique ofgene-targeting vector to multiple loci are shown as follows. First, the targeting loci were increased to 100 - 300. Second, the problems of unstable expression of inserted genes were partially solved. Third, the need for safety against toxicity integration was resolved. Fourth, the forbidden zone of gene integrating on the repetitive DNA sequences was broken through.
Animals ; Attachment Sites, Microbiological ; genetics ; Chromosomes, Artificial, Bacterial ; genetics ; Cloning, Molecular ; DNA ; genetics ; metabolism ; DNA Restriction Enzymes ; metabolism ; DNA, Ribosomal Spacer ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Integrases ; genetics ; Interferon-gamma ; genetics ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; genetics ; Transformation, Genetic

The Staphylococcus (S.) intermedius group (SIG) has been a main research subject in recent years. S. pseudintermedius causes pyoderma and otitis in companion animals as well as foodborne diseases. To prevent SIG-associated infection and disease outbreaks, identification of both staphylococcal exotoxins and staphylococcal cassette chromosome mec (SCCmec) types among SIG isolates may be helpful. In this study, it was found that a single isolate (one out of 178 SIG isolates examined) harbored the canine enterotoxin SEC gene. However, the S. intermedius exfoliative toxin gene was found in 166 SIG isolates although the S. aureus-derived exfoliative toxin genes, such as eta, etb and etd, were not detected. SCCmec typing resulted in classifying one isolate as SCCmec type IV, 41 isolates as type V (including three S. intermedius isolates), and 10 isolates as non-classifiable. Genetic relatedness of all S. pseudintermedius isolates recovered from veterinary staff, companion animals, and hospital environments was determined by pulsed-field gel electrophoresis. Strains having the same band patterns were detected in S. pseudintermedius isolates collected at 13 and 18 months, suggesting possible colonization and/or expansion of a specific S. pseudintermedius strain in a veterinary hospital.
Animals ; Bacterial Toxins/genetics/metabolism ; Cat Diseases/epidemiology/*microbiology ; Cats ; Chromosomes, Bacterial/genetics/metabolism ; Dog Diseases/epidemiology/*microbiology ; Dogs ; Electrophoresis, Gel, Pulsed-Field/veterinary ; Enterotoxins/genetics/metabolism ; Exfoliatins/genetics/metabolism ; Exotoxins/*genetics/metabolism ; Hospitals, Animal ; Humans ; Medical Staff, Hospital ; Molecular Sequence Data ; Pets/microbiology ; Polymerase Chain Reaction/veterinary ; Republic of Korea/epidemiology ; Staphylococcal Infections/epidemiology/microbiology/*veterinary ; Staphylococcus/genetics/isolation & purification ; Staphylococcus intermedius/*genetics/*isolation & purification