One step assembly of multiple DNA fragments with a designed order and orientation in Bacillus subtilis plasmid.

A universal method to reconstitute sets of genes was developed. Owing to the intrinsic nature of the plasmid establishment mechanism in Bacillus subtilis, the assembly of five antibiotic resistance genes with a defined order and orientation was achieved. These five fragments and the plasmid have three-base protruding sequences at both ends. The protruding sequences are designed so that each fragment is ligated once in a row according to the pairing. Ligation by T4 DNA ligase in the presence of 150 mM NaCl and 10% polyethylene glycol at 37 degrees C yielded high molecular tandem repeat linear form DNA. This multimeric form of DNA was preferentially used for plasmid establishment in B.subtilis. The method, referred to as Ordered Gene Assembly in B.subtilis (OGAB), allows for the design of multiple fragments with very high efficiency and great fidelity.

[1]  M Mandel,et al.  Calcium-dependent bacteriophage DNA infection. , 1970, Journal of molecular biology.

[2]  T. Trautner,et al.  Plasmid transformation in Bacillus subtilis: Effects of insertion of Bacillus subtilis DNA into plasmid pC194 , 2004, Molecular and General Genetics MGG.

[3]  Mitsuhiro Itaya,et al.  Recombinational Transfer of 100-Kilobase Genomic DNA to Plasmid in Bacillus subtilis 168 , 2001, Journal of bacteriology.

[4]  A. Paul,et al.  Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template , 2002, Science.

[5]  S. Ehrlich,et al.  Essential Bacillus subtilis genes , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[6]  B A Pfeifer,et al.  Biosynthesis of Complex Polyketides in a Metabolically Engineered Strain of E. coli , 2001, Science.

[7]  M. Itaya.  Construction of a novel tetracycline resistance gene cassette useful as a marker on the Bacillus subtilis chromosome. , 1992, Bioscience, biotechnology, and biochemistry.

[8]  H. Goodman,et al.  Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures. , 1975, Journal of molecular biology.

[9]  A. Goffeau,et al.  The complete genome sequence of the Gram-positive bacterium Bacillus subtilis , 1997, Nature.

[10]  M. Itaya,et al.  A neomycin resistance gene cassette selectable in a single copy state in the Bacillus subtilis chromosome. , 1989, Nucleic acids research.

[11]  W. Stemmer,et al.  Genome shuffling leads to rapid phenotypic improvement in bacteria , 2002, Nature.

[12]  M. Itaya,et al.  Conversion of Bacillus subtilis 168: Natto Producing Bacillus subtilis with Mosaic Genomes. , 1999, Bioscience, biotechnology, and biochemistry.

[13]  S. Horinouchi,et al.  Restriction and modification in Bacillus species , 1977, Molecular and General Genetics MGG.

[14]  M. Itaya,et al.  Efficient cloning and engineering of giant DNAs in a novel Bacillus subtilis genome vector. , 2000, Journal of biochemistry.

[15]  Richard J. Roberts,et al.  REBASE-restriction enzymes and methylases , 1997, Nucleic Acids Res..

[16]  K. Hayashi,et al.  Influence of monovalent cations on the activity of T4 DNA ligase in the presence of polyethylene glycol. , 1985, Nucleic acids research.

[17]  B. Qiang,et al.  A type II restriction endonuclease with an eight nucleotide specificity from Streptomyces fimbriatus. , 1984, Nucleic acids research.

[18]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[19]  Y. Nakamura,et al.  Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. , 2000, DNA research : an international journal for rapid publication of reports on genes and genomes.

[20]  Denis Pompon,et al.  Total biosynthesis of hydrocortisone from a simple carbon source in yeast , 2003, Nature Biotechnology.

[21]  C. Anagnostopoulos,et al.  REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS , 1961, Journal of bacteriology.

[22]  R. Schinzel,et al.  Restriction endonuclease BglI as a tool for in vitro reconstruction and recombination of plasmid and bacteriophage genomes , 2004, Molecular and General Genetics MGG.

[23]  E. Koonin,et al.  A minimal gene set for cellular life derived by comparison of complete bacterial genomes. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[24]  D. Henner.  Inducible expression of regulatory genes in Bacillus subtilis. , 1990, Methods in enzymology.

[25]  C. Harwood,et al.  Molecular biological methods for Bacillus , 1990 .

[26]  Richard J. Roberts,et al.  REBASE-restriction enzymes and methylases , 1993, Nucleic Acids Res..

[27]  T. Imanaka,et al.  Effect of in vitro DNA rearrangement in the NH2-terminal region of the penicillinase gene from Bacillus licheniformis on the mode of expression in Bacillus subtilis. , 1985, Journal of general microbiology.

[28]  K. Hayashi,et al.  Stimulation of intermolecular ligation with E. coli DNA ligase by high concentrations of monovalent cations in polyethylene glycol solutions. , 1985, Nucleic acids research.

[29]  D. Dubnau,et al.  Genetic competence in Bacillus subtilis. , 1991, Microbiological reviews.

[30]  K. Asai,et al.  A Bacillus subtilis gene‐encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition , 1998, Molecular microbiology.

[31]  T. Mizuno,et al.  Effect of increased dosage of the ML-236B (compactin) biosynthetic gene cluster on ML-236B production in Penicillium citrinum , 2002, Molecular Genetics and Genomics.