Paulina Balbás

Plasmid vector pBR322 and its special-purpose derivatives: a review

The plasmid pBR322 was one of the first EK2 multipurpose cloning vectors to be designed and constructed (ten years ago) for the efficient cloning and selection of recombinant DNA molecules in Escherichia coli. This 4363-bp DNA molecule has been extensively used as a cloning vehicle because of its simplicity and the availability of its nucleotide sequence. The widespread use of pBR322 has prompted numerous studies into its molecular structure and function. These studies revealed two features that detract from the plasmids effectiveness as a cloning vector: plasmid instability in the absence of selection and, the lack of a direct selection scheme for recombinant DNA molecules. Several vectors based on pBR322 have been constructed to overcome these limitations and to extend the vectors versatility to accommodate special cloning purposes. The objective of this review is to provide a survey of these derivative vectors and to summarize information currently available on pBR322.

Understanding the Art of Producing Protein and Nonprotein Molecules in Escherichia coli

The high-level production of functional proteins in E. coli is a very extense field of research in biotechnology. A number of important aspects to be considered in the initial design of an expression system and their interplay, were clear years ago. However, in recent times, strategies that go beyond transcription, translation, stability, vector, and strain choice, have been developed; so now expression of active peptides can be viewed as a more integrated process. Coexpression of protein subunits, foldases and chaperones, protein folding, location and purification schemes, metabolic engineering of the cell’s central metabolism, and in vitro refolding strategies, are some of the novelties that are now available to aid in the success of an efficient expression system for active heterologous proteins. This review presents a compilation of the basic issues that influence the success in the production of protein and nonprotein products in Escherichia coli, as well as some general strategies designed to facilitate downstream process operations and improve biosynthesis yields.

Carbon regulation and the role in nature of the Escherichia coli penicillin acylase (pac) gene

Quantitative analysis of specific pac mRNA and a lacZ fusion to the 5’‐terminal region of the pac gene demonstrated that both phenylacetic acid induction and catabolite repression by glucose are involved, at the transcriptional level, in the regulation of the pac gene. The studies presented here suggest that this regulation is also present in Escherichia coli transformed strains in which the pac gene was not originally present. Analysis of the nucleotide sequence of the 5′‐terminal region of this gene, with a statistical algorithm, confirms that the putative promoter previously proposed by our group is the most feasible within this region. We demonstrate that penicillin acylase activity can confer on E. coli the ability to use penicillin G as a metabolic substrate, by detaching the phenylacetic group which can be used as a carbon source. Based on these data, the regulation properties of the pac gene studied in this work, and the specificity profile of the penicillin acylase enzyme we suggest a role for it in E. coli as a scavenger enzyme for phenylacetylated compounds.

A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome

Plasmid pBRINT is an efficient vector for chromosomal integration of cloned DNA into the lacZ gene of Escherichia coli [Balbás et al., Gene 136(1993) 211-213]. A family of related plasmids containing different antibiotic-resistance markers (CmR or GmR or KmR) and a larger multiple cloning site (MCS) has been constructed. This set of plasmids, whose integration efficiencies are as good as those obtained with the prototype plasmid pBRINT, constitutes a collection of tools that allow rapid and easy integration of cloned DNA, at the chromosomal level. Their functionality as integration vectors has been ascertained by integrating the Vitreoscilla sp. hemoglobin-encoding gene and the Photobacterium leiognathi lux genes. To evaluate the level of expression obtained after chromosomal integration, we constructed strains carrying one or two copies of the cat gene integrated in the chromosome, and compared their enzymatic activities with those obtained from a strain carrying cat on a multicopy plasmid.

Chromosomal editing in Escherichia coli : Vectors for DNA integration and excision

Chromosomal editing constitutes the direct and specific modification of the genetic information present in the chromosome. In the bacterium Escherichia coli, strategies were originally developed for the production of specific proteins, the genotypic improvement of strains, and the analysis of regulation of gene expression. However, with the emerging field of metabolic engineering and genomics, efficient means of targeting specific genetic mutations into the chromosome are most useful. In this review, a summary of the systems currently available to generate insertions and deletions in the chromosome of E. coli are presented, as well as the current knowledge about the genetic mechanisms responsible for these processes.

The plasmid, pBR322.

Publisher Summary This chapter describes genetic engineering which is the transfer of DNA between hosts by in vitro enzymatic manipulations. This implies that the DNA to be transferred is duplicated in the new host. As most DNA fragments are incapable of self-replication in E. coli or any other host cell, an additional segment of DNA, capable of autonomous replication, must be linked to the fragment to be cloned. This autonomously replicating fragment is the molecular cloning vector and plays a central role in recombinant DNA technology. Most cloning vectors have been originally derived from naturally occurring extrachromosomal elements, such as bacteriophage and plasmids. Bacteriophage vectors, such as M13 and lambda, have proven to be very useful as cloning vectors. Wild-type plasmids, such as pSCl0l and ColEl have served as two of the first cloning vectors. Although both plasmids possessed certain features that made them useful as vectors, there are no vectors available at the time that possessed all of these features in one plasmid.

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 and translation, protein folding, secretion, location, posttranslational modifications, particularities of different strains, and the like and more integrated processes have been developed, the basic plasmid-borne elements and their interaction with the particular host strain will influence the overall expression system and final productivity. Plasmid vector pBR322 is a well-established multipurpose cloning vector in laboratories worldwide, and a large number of derivatives have been created for specific applications and research purposes, including gene expression in its natural host, E. coli, and few other bacteria. The early characterization of the molecule, including its nucleotide sequence, replication and maintenance mechanisms, and determination of its coding regions, accounted for its success, not only as a universal cloning vector, but also as a provider of genes and an origin of replication for other intraspecies vectors. Since the publication of the aforementioned reviews, novel discoveries pertaining to these issues have appeared in the literature that deepen the understanding of the plasmids features, behavior, and impact in gene expression systems, as well as some important strain characteristics that affect plasmid replication and stability. The objectives of this review include updating and discussing the new information about (1) the replication and maintenance of pBR322; (2) the host-related modulation mechanisms of plasmid replication; (3) the effects of growth rate on replication control, stability, and recombinant gene expression; (4) ways for plasmid amplification and elimination. Finally, (5) a summary of novel ancillary studies about pBR322 is presented.

A new expression vector for the production of fused proteins in Escherichia coli

SummaryThe construction of a new expression vector for fused proteins production in Escherichia coli is reported. This new vehicle uses the trp promoter-operator control region for the high level expression of a DNA fragment that codes for the amino terminal fragment of the cI λ repressor protein. This truncated polypeptide is accumulated as inclusion bodies that are easily purified. To probe the benefits of the system, synthetic DNA that codes for the human insulin B chain, was cloned at the end of the DNA coding region for the cI truncated peptide. The hybrid peptide thus produced after induction, allowed an easy and reproducible purification of active insulin B chain.

Plasmid pBRINT: a vector for chromosomal insertion of cloned DNA

Plasmid pBRINT is a pBR322 derivative [Bolivar et al., Gene 2 (1977) 95-113; Balbás et al., Gene 50 (1986) 3-40] that allows the insertion and replacement of DNA sequences into the Escherichia coli chromosome by homologous recombination. This method uses the inability of E. coli strain ATCC47002 (JC7623) to replicate covalently closed circular (ccc) pBR322-derived plasmids, and the convenience of XGal+IPTG screening for recombinants. The vector also contains suitable selection markers (Ap and Cm), as well as a multiple cloning site (MCS) derived from the pUC vectors [Yanisch-Perron et al., Gene 33 (1985) 103-119] to facilitate cloning. A simple PCR scheme was developed to scan for DNA insertions into the bacterial chromosome. Once introduced into the chromosome, the inserted DNA sequences can be transferred to other strains by bacteriophage P1-mediated transduction.

Production in Escherichia coli of a rat chimeric proinsulin polypeptide carrying human A and B chains and its preparative chromatography.

A pseudohuman proinsulin coding DNA sequence (MMRPI) carrying human A and B chains, was constructed via directed mutagenesis of a previously modified rat proinsulin cDNA (MRPI) and expressed as a tryptophan (Trp)LE-proinsulin fusion protein in Escherichia coli W3110. Expression of the hybrid gene was achieved by depletion of tryptophan from the medium. The heterologous fusion protein, accumulated as insoluble inclusion bodies within the cell, was obtained by differential centrifugation and then solubilized using formic acid. At the junction of the two peptides, a methionine residue allowed proinsulin to be released from the carrier protein by cyanogen bromide treatment. The sulfonated form of this proinsulin polypeptide was easily purified, at a preparative level, using ion exchange chromatography.