A totally synthetic plasmid for general cloning, gene expression and mutagenesis in Escherichia coli.

A first totally synthetic Escherichia coli plasmid has been designed, constructed and shown to be a functional, stable, high-copy cloning vector. The FokI method of gene synthesis [Mandecki and Bolling, Gene 68 (1988) 101-107] was used to assemble the plasmid from 30 oligodeoxyribonucleotides. The plasmid contains synthetic modules for the beta-lactamase-encoding gene (bla), replication origin, lacZ gene fragment and multicloning site. The plasmid is patterned after the pUC-type plasmids and has a copy number similar to that of pUC plasmids. The major changes introduced include the removal of nearly 50% of the restriction sites present in pUC plasmids, reduction of plasmid size to 2050 bp, and introduction of transcription terminators downstream from both the bla gene and lacZ fragment. The changes facilitate a number of techniques, such as cloning, mutagenesis, expression and restriction analysis.

[1]  W Mandecki,et al.  FokI method of gene synthesis. , 1988, Gene.

[2]  J. Tamm,et al.  A single base-pair alteration is responsible for the DNA overproduction phenotype of a plasmid copy-number mutant , 1981, Cell.

[3]  P. Farnham,et al.  Synthetic sites for transcription termination and a functional comparison with tryptophan operon termination sites in vitro. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[4]  G Osterburg,et al.  Nucleotide sequence of bacteriophage fd DNA. , 1978, Nucleic acids research.

[5]  J. Vieira,et al.  The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. , 1982, Gene.

[6]  G. Cesareni.  The RNA primer promoter, as defined in vitro, is essential for pMB1 plasmid replication in vivo. , 1982, Journal of molecular biology.

[7]  M. Gouy,et al.  Codon usage in bacteria: correlation with gene expressivity. , 1982, Nucleic acids research.

[8]  W Mandecki,et al.  A lac promoter with a changed distance between -10 and -35 regions. , 1982, Nucleic acids research.

[9]  J. Brosius,et al.  Precise location of two promoters for the beta-lactamase gene of pBR322. S1 mapping of ribonucleic acid isolated from Escherichia coli or synthesized in vitro. , 1982, The Journal of biological chemistry.

[10]  J. Shine,et al.  Origin of replication of pBR345 plasmid DNA. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[11]  R. Jones,et al.  In vitro evaluation of pyridine-2-azo-p-dimethylaniline cephalosporin, a new diagnostic chromogenic reagent, and comparison with nitrocefin, cephacetrile, and other beta-lactam compounds , 1982, Journal of clinical microbiology.

[12]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[13]  D. Auble,et al.  Promoter recognition by Escherichia coli RNA polymerase. Influence of DNA structure in the spacer separating the -10 and -35 regions. , 1988, Journal of molecular biology.

[14]  S. Falkow,et al.  Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. , 1977, Gene.

[15]  M. Caruthers,et al.  lac Up-promoter mutants with increased homology to the consensus promoter sequence , 1985, Journal of bacteriology.