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Copy-Control pBAC/oriV Vectors for Genomic Cloning

The use of the improved BAC system for cloning genomic DNA and library constructions is described. This system retains all the advantages of the original BACs but, in addition, permits, on command, amplification of the BAC plasmids and cloned DNA. This system consists of (1) plasmid pBAC/oriV cont ...

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Copy-Control Tightly Regulated Expression Vectors Based on pBAC/oriV

A novel type of expression vectors with a dual regulation of both the plasmid copy number and gene expression, is described. The most important and beneficial feature of these vectors is that when they are not induced, they are maintained as a single-copy plasmid, and therefore, any residual expres ...

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Cell-Free Protein Synthesis With Prokaryotic Combined Transcription-Translation

Cell-free biology exploits and studies complex biological processes in a controlled environment without intact cells. One model system is prokaryotic cell-free protein synthesis. This technology offers an attractive and convenient approach to produce properly folded recom ...

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Genetic Tools for the Manipulation of Moderately Halophilic Bacteria of the Family Halomonadaceae

Moderately halophilic bacteria of the family Halomonadaceae (Halomonas, Chromohalobacter, and Zymobacter) have promising applications in biotechnology as a source of compatible solutes (stabilizers of biomolecules and cells), salt-tolerant enzymes, biosurfactan ...

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Gene Transfer and Expression of Recombinant Proteins in Moderately Halophilic Bacteria

Moderately halophilic bacteria (MHB) of the genera Halomonas and Chromohalobacter have been used as hosts for the expression of heterologous proteins of biotechnological interest, thus expanding their potential to be used as cell factories for various applications. This chapter ...

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Recombinant Protein Production in Antarctic Gram-Negative Bacteria

This review reports some results from our laboratory on the setting up of a psychrophilic expression system for the homologous/heterologous protein production in cold-adapted bacteria by using natural plasmids as cloning vectors. By screening some Antarctic bacteria for the pres ...

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Back to Basics: pBR322 and Protein Expression Systems in E. coli

The extensive variety of plasmid-based expression systems in E. coli resulted from the fact that there is no single strategy for achieving maximal expression of every cloned gene. Although a number of strategies have been implemented to deal with problems associated to gene transcription ...

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-Complementation-Enabled T7 Expression Vectors and Their Use for the Expression of Recombinant Polypeptides for Protein Transduction Experiments

Over the past few years protein transduction has emerged as a powerful means for the delivery of proteins into cultured cells and into whole mice. This method is based on the ability of proteins containing protein transduction domains (PTDs), short stretches of 9–16 predominantly basic amino a ...

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Chromosomal Expression of Foreign and Native Genes From Regulatable Promoters in Escherichia coli

A two-step cloning system for expression of foreign and native genes from heterologous promoters in single copy from the E. coli chromosome is described. The system is based on the conditional-replication integration and modular CRIM plasmid technology and new CRIM plasmids described h ...

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Overexpression of Chromosomal Genes in Escherichia coli

Conversion of some carbon sources into desired compounds by a biological system is the goal of many biotechnologists. The understanding of the mechanisms by which an organism does these conversions permits the improved production of specific metabolites. Pathway engineering invo ...

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Expression of Recombinant Alkaline Phosphatase Conjugates in Escherichia coli

The methods described in this article are relative to the use of a positive cloning/screening recombinant system for the generation in Escherichia coli of foreign proteins fused to a highly active bacterial alkaline phosphatase (PhoA) variant as reporter enzyme. Appropriate insert ...

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Controlled Expression of Homologous Genes by Genomic Promoter Replacement in the Yeast Saccharomyces cerevisiae

Exchange of the promoter of a gene in the genome for another promoter whose expression can be controlled easily can overcome problems associated with the expression of the same gene from a promoter on a plasmid. Some genes are difficult or impossible to clone in plasmid-based vectors and often a stab ...

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High-Throughput Expression in Microplate Format in Pichia pastoris

The methylotrophic yeast Pichia pastoris has become a powerful host for the heterologous expression of proteins. To serve the increasing demand for clones expressing different cDNAs, we developed a cultivation and induction protocol amenable to automation to increase the number of c ...

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High-Throughput Expression in Microplate Format in Saccharomyces cerevisiae

We have developed a high-throughput technology that allows parallel expression, purification, and analysis of large numbers of cloned cDNAs in the yeast Saccharomyces cerevisiae. The technology is based on a vector for intracellular protein expression under control of the inducib ...

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Multiple Gene Expression by Chromosomal Integration and CRE-loxP-Mediated Marker Recycling in Saccharomyces cerevisiae

Multiple gene expression can be introduced in a yeast strain with using only two markers by means of the two new vectors described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette con ...

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Three Decades of Fungal Transformation: Key Concepts and Applications

Filamentous fungi include a large, heterogeneous group of heterotrophic organisms with profound influence in human activities. In spite of their economic and scientific relevance, little information is available at the molecular level about their biology. The development of gen ...

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Three Decades of Fungal Transformation: Novel Technologies

Fungi are lower eukaryotes that play important roles in many human activities, including biotechnological processes, phytopathology, and biomedical research. In addition, they are excellent models for molecular and genetic studies. An important key in the advancement of geneti ...

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Gene Transfer and Expression in Plants

Until recently, agriculture and plant breeding relied solely on the accumulated experience of generations of farmers and breeders—that is, on sexual transfer of genes between plant species. However, recent developments in plant molecular biology and genomics now give us access to kno ...

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Production of Recombinant Proteins by Hairy Roots Cultured in Plastic Sleeve Bioreactors

Plant-based expression of recombinant proteins offers significant advantages over transgenic animal-and cell-based systems. Unlike bacteria, plants perform the complex protein-processing steps required to produce eukaryotic proteins in active form. In order to facili ...

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Engineering the Chloroplast Genome for Hyperexpression of Human Therapeutic Proteins and Vaccine Antigens

The chloroplast genome is ideal for engineering because it offers a number of attractive advantages, including high-level gene expression, the feasibility of expressing multiple genes or pathways in a single transformation event, and transgene containment due to lack of pollen tran ...

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