Inducible gene expression systems are vital tools for the advancement of synthetic biology. Their application as genetically encoded biosensors has the potential to contribute to diagnostics and to revolutionise the field of microbial cell factory development. Currently, the number of compounds of biological interest by far exceeds the number of available biosensors. Here, we address this limitation by developing a generic genome-wide approach to identify transcription factor-based inducible gene expression systems. We construct and validate 15 functional biosensors, provide a characterisation workflow to facilitate forward engineering efforts, exemplify their broad-host-range applicability, and demonstrate their utility in enzyme screening. Previously uncharacterised interactions between sensors and compounds of biological relevance are identified by employing the largest reported library of metabolite-responsive biosensors in an automated high-throughput screen. With the rapidly growing genomic data these innovative capabilities offer a platform to vastly increase the number of biologically detectable molecules.

Infectious cDNA clones are an important tool to study the molecular and cellular process of RNA virus infection. In vitro and in vivo transcription systems are the two main strategies used in the generation of infectious cDNA clones for RNA viruses. This study describes the first generation of a full-length infectious cDNA clone of Cowpea mild mottle virus (CPMMV), a Carlavirus. The full-length genome was synthesized by Overlap Extension PCR of two overlapping fragments and cloned in a pUC-based vector under control of the SP6 RNA polymerase promoter. After in vitro run-off transcription, the produced RNA was mechanically inoculated into soybean plants cv. CD206. The systemic infection was confirmed by RT-PCR and further sequencing of amplified cDNA fragments. To simplify the transfection process, the complete genome was subcloned into a binary vector under control of the 35S promoter of cauliflower mosaic virus by the Gibson Assembly protocol. The resulting clones were inoculated by particle bombardment onto soybean seedlings and the recovery of the virus was confirmed 2 weeks later by RT-PCR. Our results indicate the constructs of the full-length cDNA of CPMMV are fully infectious in both in vitro and in vivo transcription strategies.

No abstract

RESUMOIntrodução: Biomarcadores vem sendo utilizados para monitorar o uso do álcool e, atualmente, os mais sensíveis e específicos são enzimas hepáticas, por exemplo, gama glutamiltransferase (GGT), alanina aminotransferase (ALT), aspartato aminotransferase (AST) e fosfatase alcalina (ALP). Objetivo: Verificar, a partir da experimentação animal, as alterações provocadas pelo uso de álcool e pela prática de atividade física nas enzimas hepáticas e pancreáticas. Métodos: Vinte e quatro ratos da linhagem Wistar foram distribuídos aleatoriamente em grupos experimentais, alojados em gaiolas com ambiente controlado, divididos de acordo com os tratamentos recebidos. No tratamento inicial, foi administrado álcool aos grupos álcool sedentário (AS) e álcool exercitado (AE) e, ao final da quarta semana, iniciou-se o programa de treinamento físico em esteira com os grupos AE e controle exercitado (CE). A coleta de sangue foi realizada por punção cardíaca ao final de cada experimento. Na análise estatística, utilizou-se teste de análise de variância (ANOVA) seguido de teste de Tukey e teste de Kruskal-Wallis. Resultados: O grupo AS apresentou valores significativamente mais elevados de ALT e ALP quando comparado aos grupos CE e AE, respectivamente. Não foram observadas diferenças significativas entre os quatro grupos estudados para os parâmetros AST, GGT e amilase. Conclusão: A associação entre consumo de álcool e sedentarismo aumentou a liberação das enzimas ALT e ALP em ratos Wistar; a prática de exercício físico aeróbico após abstinência alcoólica evitou o aumento da liberação de ALP no plasma desses animais.Descritores: alcoolismo/enzimologia, atividade motora, ratos Wistar. ABSTRACT Introduction: Biomarkers have been used to monitor the use of alcohol and currently the most sensitive and specific are the liver enzymes, for example, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP

No abstract