RESUMEN
Vector construction and gene cloning are ubiquitous techniques essential to all fields of biological and medical research. They are the first steps in many endeavors leading to expressing proteins to understand gene function and regulation. However, they can often be rate-limiting, particularly in multi-gene studies, due to the time and effort required to assemble gene constructs and to identify the optimal constructs for protein expression.The SureVector system was developed to address this by enabling the rapid and reliable assembly of multiple DNA modules into a recombinant plasmid containing a gene-of-interest (GOI). It harnesses the power of synthetic biology to combine DNA modules from standard parts into a customized vector that expresses proteins in bacterial, mammalian, or yeast cells. The key advantages of the innovative SureVector system include rapid custom vector generation, enhanced flexibility to assemble new vectors quickly as experimental requirements change, and the reliable and precise assembly of fully interchangeable standard DNA modules that retain their functionality. The SureVector system is the only next-generation plasmid assembly technology to guarantee assembly of multiple functional DNA modules.
Asunto(s)
Eucariontes/genética , Células Eucariotas/metabolismo , Expresión Génica/genética , Vectores Genéticos/genética , Células Procariotas/metabolismo , Proteínas/genética , Animales , Bacterias/genética , Clonación Molecular/métodos , ADN/genética , Mamíferos/genética , Plásmidos/genética , Recombinación Genética/genética , Biología Sintética/métodos , Levaduras/genéticaRESUMEN
Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd ) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.
Asunto(s)
Bacillus anthracis/genética , Bacillus anthracis/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Animales , Carbunco/diagnóstico , Carbunco/microbiología , Bacillus anthracis/clasificación , Bioterrorismo , Monitoreo del Ambiente , Genes Bacterianos , Genotipo , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The complete nucleotide sequences of eight West Nile (WN) virus strains (Egypt 1951, Romania 1996-MQ, Italy 1998-equine, New York 1999-equine, MD 2000-crow265, NJ 2000MQ5488, NY 2000-grouse3282, and NY 2000-crow3356) were determined. Phylogenetic trees were constructed from the aligned nucleotide sequences of these eight viruses along with all other previously published complete WN virus genome sequences. The phylogenetic trees revealed the presence of two genetic lineages of WN viruses. Lineage 1 WN viruses have been isolated from the northeastern United States, Europe, Israel, Africa, India, Russia, and Australia. Lineage 2 WN viruses have been isolated only in sub-Saharan Africa and Madagascar. Lineage 1 viruses can be further subdivided into three monophyletic clades.
