RESUMO
Gene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. Here, we describe a high-throughput platform to design and produce multiple synthetic genes (<500 bp) for recombinant expression in Escherichia coli. This pipeline includes an innovative codon optimization algorithm that designs DNA sequences to maximize heterologous protein production in different hosts. The platform is based on a simple gene synthesis method that uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides. This technology incorporates an accurate, automated and cost-effective ligase-independent cloning step to directly integrate the synthetic genes into an effective E. coli expression vector. High-throughput production of synthetic genes is of increasing relevance to allow exploring the biological function of the extensive genomic and meta-genomic information currently available from various sources.
Assuntos
Genes Sintéticos/genética , Ensaios de Triagem em Larga Escala/métodos , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/genética , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica/genética , Proteínas Recombinantes/biossínteseRESUMO
Escherichia coli is a common host for recombinant protein production in which production titers are highly dependent on the employed expression system. Promoters are thereby a key element to control gene expression levels. In this study, a novel PLICable promoter toolbox was developed which enables in a single cloning step and after a screening experiment to identify out of ten IPTG-inducible promoters (T7, A3, lpp, tac, pac, Sp6, lac, npr, trc and syn) the most suitable one for high level protein production. The target gene is cloned under the control of different promoters in a single and efficient cloning step using the ligase-free cloning method PLICing (phosphorothioate-based ligase-independent gene cloning). The promoter toolbox was firstly validated using three well producible proteins (a cellulase from a metagenome library, a phytase from Yersinia mollaretii and an alcohol dehydrogenase from Pseudomonas putida) and then applied to two enzymes (3D1 DNA polymerase and glutamate dehydrogenase mutant) which are poorly produced in E. coli. By applying our PLICable pET-promoter toolbox, the authors were able to increase production by two-fold for 3D1 DNA polymerase (lac promoter) and 29-fold for glutamate dehydrogenase mutant H52Y (trc promoter).
Assuntos
Escherichia coli/genética , Regiões Promotoras Genéticas , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , 6-Fitase/genética , 6-Fitase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Celulase/genética , Celulase/metabolismo , Escherichia coli/metabolismo , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Recombinantes/genética , Reprodutibilidade dos TestesRESUMO
Yeast one-hybrid (Y1H) assay has been proven to be a powerful technique to characterize in vivo the interaction between a given transcription factor (TF), or its DNA-binding domain (DBD), and target DNA sequences. Comprehensive characterization of TF/DBD and DNA interactions should allow designing synthetic promoters that would undoubtedly be valuable for biotechnological approaches. Here, we use the ligation-independent cloning system (LIC) in order to enhance the cloning efficiency of DNA motifs into the pHISi Y1H vector. LIC overcomes important limitations of traditional cloning technologies, since any DNA fragment can be cloned into LIC compatible vectors without using restriction endonucleases, ligation, or in vitro recombination.
Assuntos
Clonagem Molecular/métodos , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Sequência de Bases , Regulação da Expressão Gênica , Vetores Genéticos , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genéticaRESUMO
Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating "smart" libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field.
Assuntos
Bioengenharia , Evolução Molecular Direcionada , Variação Genética , Animais , Clonagem Molecular , Biblioteca Gênica , Humanos , Camundongos , Mutagênese Sítio-DirigidaRESUMO
Background: As key gene regulators, microRNAs post-transcriptionally modulate gene expression via binding to partially complementary sequence in the 3' UTR of target mRNA. An accurate, rapid and quantitative tool for sensing and validation of miRNA targets is of crucial significance to decipher the functional implications of miRNAs in cellular pathways. Results: Taking advantage of an improved restriction-free cloning method, we engineered a novel built-in dual luciferase reporter plasmid where Firefly and Renilla luciferase genes were assembled in a single plasmid named pFila. This design eliminates the transfection of a separate control plasmid and thus minimizes the time and labor required for miRNA-target sensing assays. pFila consistently produces Firefly and Renilla luciferase activities when transfected into human-, monkey- and mouse-derived mammalian cell systems. Moreover, pFila is capable of recapitulating the interaction of miR-16 and its known target CCNE1 in Hela cells. Additionally, pFila is shown to be a sensitive miR-biosensor by evaluating the inhibition efficiency of endogenous miRNA. Conclusions: pFila would facilitate miRNA target identification and verification in a rapid and simplified manner. Also, pFila is a sensitive biosensor for active miRNA profiling in vivo.
