isopropyl-thiogalactoside and 5-bromo-4-chloro-3-indolyl-beta-galactoside

Many plasmid vectors (e.g., the pUC series, Bluescript, pGem, and their derivatives) carry a short segment of

Topics: Colony Count, Microbial; Escherichia coli; Galactosides; Indoles; Isopropyl Thiogalactoside; Plasmids; Recombination, Genetic

Although blue-white screening based on α-complementation has been widely used in the screening of genetically modified bacteria, only a single blue-white screening is typically not enough to eliminate false positives. Sometimes, a secondary blue-white screening for the target colonies is required. In this study, two methods were used to investigate the feasibility of secondary blue-white screening for target colonies on lysogeny broth (LB)-ampicillin agar plates. The first method consisted of covering the target colonies grown on LB-ampicillin plate medium with a sterilized filter paper soaked in a solution of 60 μL 20 mg/mL X-gal and 8 μL 20% IPTG. The second method was that blue and white colonies were randomly selected from the blue-white screening plate medium and then re-streaked onto the blue-white screening medium. The colonies were then treated by two methods and incubated at 37°C for 12 h. The results showed that some of the white colonies treated by the two methods showed results similar to the colonies grown on the blue-white screening medium. These results indicate that the target colonies grown on blue-white screening medium can still be used to carry out a secondary blue-white screening. Thus, a blue-white screening liquid was successfully developed. Using the blue-white screening liquid, false positives can be eliminated directly based on the color of the target colonies. This will greatly improve the screening efficiency of positive clones and has important practical implications.

Topics: Agar; Bacteria; Colony Count, Microbial; Culture Media; Galactosides; Genetic Engineering; Indoles; Isopropyl Thiogalactoside

Shotgun sequencing and assembly of a large, complex genome can be both expensive and challenging to accurately reconstruct the true genome sequence. Repetitive DNA arrays, paralogous sequences, polyploidy, and heterozygosity are main factors that plague de novo genome sequencing projects that typically result in highly fragmented assemblies and are difficult to extract biological meaning. Targeted, sub-genomic sequencing offers complexity reduction by removing distal segments of the genome and a systematic mechanism for exploring prioritized genomic content through BAC sequencing. If one isolates and sequences the genome fraction that encodes the relevant biological information, then it is possible to reduce overall sequencing costs and efforts that target a genomic segment. This chapter describes the sub-genome assembly protocol for an organism based upon a BAC tiling path derived from a genome-scale physical map or from fine mapping using BACs to target sub-genomic regions. Methods that are described include BAC isolation and mapping, DNA sequencing, and sequence assembly.

Topics: Chromosomes, Artificial, Bacterial; Contig Mapping; DNA Fingerprinting; DNA Restriction Enzymes; DNA, Bacterial; Escherichia coli; Galactosides; Genome, Bacterial; Genomic Library; High-Throughput Nucleotide Sequencing; Indoles; Isopropyl Thiogalactoside; Sequence Analysis, DNA; Software

The genomic DNA libraries based on Bacteria Artificial Chromosomes (BAC) are the foundation of whole genomic mapping, sequencing, and annotation for many species like mice and humans. With their large insert size, BACs harbor the gene-of-interest and nearby transcriptional regulatory elements necessary to direct the expression of the gene-of-interest in a temporal and cell-type specific manner. When replacing a gene-of-interest with a transgene in vivo, the transgene can be expressed with the same patterns and machinery as that of the endogenous gene. This chapter describes in detail a method of using lambda-red recombineering to make BAC transgene constructs with the integration of a transgene into a designated location within a BAC. As the final BAC construct will be used for transfection in cell lines or making transgenic animals, specific considerations with BAC transgenes such as genotyping, BAC coverage and integrity as well as quality of BAC DNA will be addressed. Not only does this approach provide a practical and effective way to modify large DNA constructs, the same recombineering principles can apply to smaller high copy plasmids as well as to chromosome engineering.

Topics: Animals; Animals, Genetically Modified; Bacteriophage lambda; Cell Line; Chromosomes, Artificial, Bacterial; DNA, Bacterial; Electroporation; Escherichia coli; Galactosides; Genetic Engineering; Genomic Library; Homologous Recombination; Indoles; Isopropyl Thiogalactoside; Operon; Plasmids; Transgenes

Using published plasmid vectors containing the bgaB gene encoding a heat-stable beta-galactosidase, we have been unable to fuse strong promoters to this reporter gene. In addition, we could not analyze the promoter strength by a plate assay. Therefore, we constructed an Escherichia coli-Bacillus subtilis shuttle vector to allow the cloning of strong promoters and their rapid analysis in B. subtilis by plating on X-Gal plates in the presence of the inducer IPTG. We show that the blue color of the colonies reflects the strength of the promoters, which was verified by measuring the beta-galactosidase activities.

Topics: Bacillus subtilis; beta-Galactosidase; Escherichia coli; Galactosides; Genes, Reporter; Genetic Vectors; Hot Temperature; Indoles; Isopropyl Thiogalactoside; Regulatory Sequences, Nucleic Acid

A solid agar plate assay was devised to discriminate bacteriolytic from bacteriostatic activity for a given antibacterial agent. The assay uses a bacterial culture harboring beta-galactosidase enzyme as reporter of cellular lysis. When a drop of bacteriolytic compound is placed on the agar, beta-galactosidase is released from the bacteria to the external solid medium where it hydrolyzes X-Gal substrate analogue, developing a blue halo at the edge of the inhibition growth zone. The assay was successfully evaluated against several antibiotics with well-known mechanism of action. It was found that bacteriostatic compounds consistently did not display blue halo at the inhibition zone.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteriolysis; beta-Galactosidase; Chromogenic Compounds; Enterobacteriaceae; Erythromycin; Galactosides; Hydrolysis; Indoles; Isopropyl Thiogalactoside; Microbial Sensitivity Tests; Penicillins; Peptides; Reproducibility of Results; Tetracycline

For the past decade the immune system has been exploited as a rich source of de novo catalysts. Catalytic antibodies have been shown to have chemoselectivity, enantioselectivity, large rate accelerations, and even an ability to reroute chemical reactions. In many instances catalysts have been made for reactions for which there are no known natural or man-made enzymes. Yet, the full power of this combinatorial system can only be exploited if there was a system that allows for the direct selection of a particular function. A method that allows for the direct chemical selection for catalysis from antibody libraries was so devised, whereby the positive aspects of hybridoma technology were preserved and re-formatted in the filamentous phage system to allow direct selection of catalysis. This methodology is based on a purely chemical selection process, making it more general than biologically based selection systems because it is not limited to reaction products that perturb cellular machinery.

Topics: Animals; Antibodies, Catalytic; beta-Galactosidase; Catalysis; Cloning, Molecular; Coliphages; Dithiothreitol; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Galactosides; Haptens; Hybridomas; Immunoglobulin Fab Fragments; Indoles; Isopropyl Thiogalactoside; Mice; Nitrophenylgalactosides; Peptide Library; Polymerase Chain Reaction; Serum Albumin, Bovine; Transformation, Bacterial

Topics: Cell Line; Exons; Galactosides; Humans; Indoles; Isopropyl Thiogalactoside; Manganese; Melanocyte-Stimulating Hormones; Mutagenesis; Nucleotides; Point Mutation; Polymerase Chain Reaction; Sequence Analysis, DNA

Topics: Bacteriophage M13; Culture Media; Galactosides; Gene Library; Indoles; Isopropyl Thiogalactoside; Lac Operon; Virus Cultivation

An improved method for coliphage detection based on the induction of beta-galactosidase in Escherichia coli is described. Upon infection by coliphages, the cells are lysed and a stable indolyl product that is dark blue becomes visible within each plaque. The improved method is compared to the proposed coliphage detection procedure described in Standard Methods for the Examination of Water and Wastewater.

Topics: beta-Galactosidase; Coliphages; Enzyme Induction; Escherichia coli; Galactosides; Indoles; Isopropyl Thiogalactoside; Viral Plaque Assay; Water Microbiology