RESUMEN
A cell-free system for preparative gene expression is described. It is composed of DNA-free Escherichia coli extract and added plasmid DNA; coupled transcription-translation proceeds with a continuous flow of the feeding solution containing nucleoside triphosphates and amino acids. The system works at a high constant rate for tens of hours. The yield of synthesised proteins after 20-50 h is hundreds of micrograms from 1 ml of the reaction mixture. Electrophoretic analysis of translation products confirms synthesis of proteins of the expected molecular mass.
Asunto(s)
Escherichia coli/genética , Expresión Génica , Adenosina Trifosfato/metabolismo , Tampones (Química) , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Guanosina Trifosfato/metabolismo , Cinética , Nucleótidos , Plásmidos , Biosíntesis de Proteínas , Tetrahidrofolato Deshidrogenasa/genética , Transcripción Genética , beta-Lactamasas/biosíntesis , beta-Lactamasas/genéticaRESUMEN
A system is described in which manipulations of nucleic acids are performed in wells containing predispensed lyophilized reaction mixtures requiring addition of only nucleic acid. This allows increased reproducibility for single-step reactions (e.g., restrictions and ligations), as well as improved productivity for complex reactions (e.g., sequencing). Enzymes, co-factors, nucleotides and buffers can be dried and stored at room temperature without loss of essential function. When used for DNA sequencing, hundreds of templates a day can be sequenced with the potential to determine megabase amounts of sequence per week.
Asunto(s)
Ácidos Nucleicos , Secuencia de Bases , Biotecnología , ADN , ADN Ligasas , Enzimas de Restricción del ADN , Liofilización , Indicadores y ReactivosRESUMEN
This paper describes the construction of a new plasmid and M13 phage cloning vector in which the 54-bp polylinkers of pUC19 and of M13mp8 are replaced with a 45-bp chemically synthesized polylinker with different restriction sites. The new polylinker is inserted in frame at the N-terminal end of the beta galactosidase lac Z fragment. The plasmid was named pLH1, the phage M13LH1.
Asunto(s)
Vectores Genéticos , Bacteriófagos/genética , Secuencia de Bases , Biotecnología , Clonación Molecular/métodos , ADN/genética , Enzimas de Restricción del ADN , Datos de Secuencia Molecular , PlásmidosRESUMEN
A streamlined, reproducible boiling method for preparing plasmid as well as phage replicative form DNA is described. Both quantity and quality of DNA purified are sufficient for restriction enzyme analysis and sequencing. A small loopful of bacteria will provide enough DNA for several experiments, and multiple samples can be processed and prepared for digestion or sequencing in under 20 minutes. DNA preparations are stable for at least 6 months at -20 degrees C.
Asunto(s)
ADN Viral/aislamiento & purificación , Calor , Plásmidos , Bacterias/genética , Bacteriófagos/genética , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Mapeo RestrictivoRESUMEN
A heatstable alpha amylase gene was shotgun cloned from Bacillus licheniformis RPO1 into Bacillus subtilis. Restriction endonuclease analysis of the recombinant plasmid revealed a map which was identical to a previously cloned alpha amylase from B. licheniformis FDO2 and very similar to the restriction map of a high temperature amylase from Bacillus coagulans. The thermostability and temperature optimum of the cloned alpha amylase was measureably different from those of the previously reported cloned alpha amylases.
Asunto(s)
Bacillus subtilis/enzimología , Clonación Molecular , alfa-Amilasas/genética , Bacillus subtilis/genética , Enzimas de Restricción del ADN , ADN Bacteriano , Calor , PlásmidosRESUMEN
The DNA sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene is reported. Comparison of the inferred amino acid sequence of the B. licheniformis alpha-amylase gene with that of the Bacillus amyloliquefaciens gene shows that whereas the amino acid sequences of the mature proteins have considerable homology, the sequences for the signal peptides are distinct.
Asunto(s)
Bacillus/genética , Genes Bacterianos , Genes Reguladores , Péptidos/genética , alfa-Amilasas/genética , Secuencia de Aminoácidos , Bacillus/enzimología , Secuencia de Bases , ADN Bacteriano , Operón , Señales de Clasificación de ProteínaRESUMEN
A resident-plasmid cloning system developed for Bacillus subtilis has been used to isolate recombinant plasmids carrying DNA from Bacillus licheniformis which confer alpha-amylase activity on alpha-amylase-negative mutants of B. subtilis. These plasmids contain a 3550-bp insert at the EcoRI site of the plasmid pBD64. Subcloning various lengths of the B. licheniformis DNA has localised the gene to a 2550-bp BclI fragment. We present evidence that the cloned fragment codes for a B. licheniformis heat-stable alpha-amylase with a temperature optimum of 93 degrees C. The foreign gene is expressed efficiently in B. subtilis and is stably maintained.