A rapid reporter system using GFP as a reporter protein for identification and screening of synthetic stationary-phase promoters in Escherichia coli

To develop a rapid reporter system for the screening of stationary-phase promoters in Escherichia coli, the expression pattern of the green fluorescent protein (GFP) during bacterial cultivation was compared with that of the commonly used β-galactosidase. Using GFP with enhanced fluorescence, the expression pattern of both reporter systems GFP and β-galactosidase were similar and showed a typical induction of gene activity of the reporter genes, i.e. increase of expression at the transition from exponential to stationary phase. The expression was affected by the culture medium, i.e. in contrast to the complex medium (LB medium), the stationary-phase specific induction was only observed in synthetic medium (M9) when amino acids were added, whereas there was generally no induction in MOPS medium. To develop a rapid screening method on agar plates for stationary-phase promoters, a photographic approach was used, continued with computational image treatment. A screening method is presented which enables an on-line monitoring of gene activity.     

Analysis of splice donor and acceptor site function in a transposable gene trap derived from the maize element Activator

Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3′ Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.     

An advanced blue-white screening method for construction of shRNA expression vectors

Short hairpin RNA (shRNA) encoded within an expression vector is an effective tool for exploration of gene function in mammalian cells. Many of the current methods for constructing shRNA expression vectors require cumbersome and time-consuming procedures for identification of the desired recombinants. We have developed a highly efficient and less labor-intensive cloning method that allows the construction of shRNA expression vectors in one day and with minimal effort. This advanced blue-white screening technique was developed by combining the reconstitution of ideal lacO with TA cloning. The DNAs are simply ligated into the destination vectors and, following transformation, a desired recombinant event will give a typical blue colony. In addition, we have used this cloning method for the construction of targeting reporter expression vectors to measure the efficacy of the corresponding shRNA. We constructed 122 functional shRNA expression vectors and sequencing of the positive cloning vectors confirmed a high degree of accuracy. Only three short DNA primers are needed for constructing both shRNA and targeting reporter expression vectors. This advanced blue-white screening system is a powerful tool for the high-throughput assay of RNAi libraries.     

Analysis of splice donor and acceptor site function in a transposable gene trap derived from the maize element Activator

Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3 Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.     

pLR: A lentiviral backbone series to stable transduction of bicistronic genes and exchange of promoters

Gene transfer based on lentiviral vectors allow the integration of exogenous genes into the genome of a target cell, turning these vectors into one of the most used methods for stable transgene expression in mammalian cells, in vitro and in vivo. Currently, there are no lentivectors that allow the cloning of different genes to be regulated by different promoters. Also, there are none that permit the analysis of the expression through an IRES (internal ribosome entry site) - reporter gene system. In this work, we have generated a series of lentivectors containing: (1) a malleable structure to allow the cloning of different target genes in a multicloning site (mcs); (2) unique site to exchange promoters, and (3) IRES followed by one of two reporter genes: eGFP or DsRed. The series of the produced vectors were named pLR (for lentivirus and RSV promoter) and were fairly efficient with a strong fluorescence of the reporter genes in direct transfection and viral transduction experiments. This being said, the pLR series have been found to be powerful biotechnological tools for stable gene transfer and expression.     

Vector-Dependent Gene Expression Driven by Insulated P-Element Reporter Vectors

The Pelican and Stinger series of P-element transformation vectors are a popular choice for reporter gene expression in transgenic flies. We report here as a cautionary note that these vectors on their own can drive reporter gene expression in the larval and pupal salivary gland.     

Genetic and biochemical characterization of the red gene cluster of Streptomyces coelicolor A3(2)

Production of the red antibiotic, undecylprodigiosin, by Streptomyces coelicolor A3(2) was studied by DNA cloning and biochemical analysis. Over 21 kb of genomic DNA were cloned, in several segments, into plasmid vectors. The cloned DNA 'complemented' several specific mutations in the red gene cluster. Four red genes (redA, B, E, and F) were mapped to different regions within the cloned DNA. Screening with redE probes for DNA homologies among various streptomycetes revealed hybridizing DNA in three strains, one of them not known to synthesize prodigiosin pigments. Biochemical studies using protoplasted cells revised our interpretation of the nature of redE and redF mutations. Two forms of undecylnorprodigiosin: S-adenosylmethionine O-methyltransferase activity on gel filtration columns were detected: a very high molecular mass peak (greater than 5 MDal) and a 49 kDal) and a 49 kDal peak. Analyses of extracts from red mutants suggested that these two forms are related, and that at least the redE and redF gene products are necessary for O-methyltransferase activity in vivo. Lack of activity of the redE gene in a heterologous host, S. glaucescens, is consistent with the necessity for a biosynthetic complex involving several red gene products for efficient expression. Experiments in liquid antibiotic production medium indicated that prodigiosin compounds in S. coelicolor are examples of 'secondary metabolites' whose synthesis lags behind that of cell mass. The peak of specific activity of O-methyltransferase coincided with the 'late exponential' phase of growth. Thus, understanding the genetic regulation of undecylprodigiosin biosynthesis in S. coelicolor may be relevant to other antibiotic production pathways, and perhaps to 'secondary' metabolism in general.     

Vector-dependent gene expression driven by insulated P-element reporter vectors

The Pelican and Stinger series of P-element transformation vectors are a popular choice for reporter gene expression in transgenic flies. We report here as a cautionary note that these vectors on their own can drive reporter gene expression in the larval and pupal salivary gland.     

The Cre-<Emphasis Type="Italic">loxP</Emphasis> recombination-based reporter system for plant transcriptional expression studies*

To facilitate the characterization of plant genes, the Cre-loxP site-specific recombination system was adapted to make reporter vectors for plant expression studies. This system allows promoter fragments to be cloned into a small vector (univector) and subsequently recombined in vitro with binary vectors containing different reporter genes precisely at near-perfect efficiency. We have constructed univector-adapted vectors with three reporters, beta-glucuronidase, luciferase, and green fluorescent protein, and a BASTA-resistance gene for selection of plant transformants. Expression in plants using the new system was validated by comparison to conventional reporter vectors. These new vectors are efficient and economical alternatives to the other plant reporter vectors currently available. The royalty-free Cre-loxP system serves as a platform for the future expansion of recombination-based cloning vectors for plant research.