RESUMO
Minimal plasmids play an essential role in many intermediate steps in molecular biology. For example, they can be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185-bp fully functional high-copy cloning plasmid with an extended multiple cloning site. We believe that this is the smallest high-copy cloning vector ever described.
Assuntos
Clonagem Molecular/métodos , Escherichia coli/genética , Vetores Genéticos/genética , Plasmídeos/genética , Sequência de Bases , Mutagênese , Reação em Cadeia da Polimerase/métodos , Recombinação Genética , Deleção de Sequência , Biologia Sintética/métodosRESUMO
In contrast to most common gene delivery techniques, lentiviral vectors allow targeting of almost any mammalian cell type, even non-dividing cells, and they stably integrate in the genome. Therefore, these vectors are a very powerful tool for biomedical research. Here we report the generation of a versatile new set of 22 lentiviral vectors with broad applicability in multiple research areas. In contrast to previous systems, our platform provides a choice between constitutive and/or conditional expression and six different C-terminal fusions. Furthermore, two compatible selection markers enable the easy derivation of stable cell lines co-expressing differently tagged transgenes in a constitutive or inducible manner. We show that all of the vector features are functional and that they contribute to transgene overexpression in proof-of-principle experiments.
Assuntos
Engenharia Genética/métodos , Vetores Genéticos/genética , Lentivirus/genética , Proteínas Recombinantes/genética , Transgenes/genética , Transdução GenéticaRESUMO
Candida bombicola is a yeast species known to synthesize glycolipids. Although these glycolipids find several industrial, cosmetic and pharmaceutical applications, very little is known about the genetics of C. bombicola. A basic tool for genetic study and modification is the availability of an efficient transformation and selection system. In order to develop such a system, the URA3 gene of Candida bombicola was isolated using degenerate PCR and genomic walking. The gene encodes for an enzyme of 262 amino acids and shows high homology with the known orotidine-5'-phosphate decarboxylases of several other yeast species. The functionality of the gene was proved by complementation of a URA3-negative Saccharomyces cerevisiae strain.