遗传学实验技术

The web application PrimerPair at ecogene.org generates large sets of paired DNA sequences surrounding� all protein and RNA genes of Escherichia coli K-12. Many DNA fragments, which these primers amplify, can be used to implement a genome reengineering strategy using complementary in vi ...

A protocol is described that allows the transfer of genetic material from one Escherichia coli strain to another using bacteriophage P1. P1 transduction can be used to construct new bacterial strains containing multiple alleles, to restore a locus to wild type, to move specific genetic mark ...

An improved and rapid genomic engineering method has been developed for the construction of �custom-designed microorganisms by scarless chromosomal gene knockouts. This method, which can be performed in 2 days, permits restructuring of the Escherichia coli genome via scarless del ...

The λ phage Red proteins greatly enhance homologous recombination in Escherichia coli. Red-mediated recombination or “recombineering” can be used to construct targeted gene deletions as well as to introduce point mutations into the genome. Here, we describe our method for scanning mu ...

Strain engineering of bacteria has been accomplished by many methods where mobile DNA elements (transposons) are inserted into the genomic DNA of a host organism. This chapter addresses engineering with transposable elements complexed with transposase enzyme. In traditional te ...

Successful strain engineering involves perturbing key nodes within the cellular network. How the �network’s connectivity affects the phenotype of interest and the ideal nodes to modulate, however, are frequently not readily apparent. To guide the generation of a list of candidate nod ...

Constructing polycistronic operons is an advantageous strategy for coordinating the expression of �multiple genes in a prokaryotic host. Unfortunately, a basic construct consisting of an inducible promoter and genes cloned in series does not generally lead to optimal results. He ...

Genetic manipulation of Escherichia coli strains for desired traits is the most applied strain engineering approach in industrial applications. For chromosomal insertion of genes and controlled expression of genomic genes in E. coli, the replicon-free and markerless method is des ...

Saccharomyces cerevisiae, commonly known as baker’s or budding yeast, is an attractive organism for design-based engineering because it is an industrially important organism with a well-annotated genome sequence and an extensive collection of resources for molecular analyses. ...

Gene inactivation is an essential step in the molecular dissection of gene function. In the yeast Saccharomyces cerevisiae, many tools for gene disruption are available. Gene disruption cassettes comprising completely heterologous marker genes flanked by short DNA segments hom ...

Transposon mutagenesis is an effective method for generating large sets of random mutations in target DNA, with applicability toward numerous types of genetic screens in prokaryotes, single-celled eukaryotes, and metazoans alike. Relative to methods of random mutagenesis by che ...

The availability of collections of genome-wide deletion mutants greatly accelerates systematic analyses of gene function. However, each of the thousands of genes that comprise a genome must be phenotyped individually unless they can be assayed in parallel and subsequently deconv ...

Cellular hosts are widely used for the production of chemical compounds including pharmaceutics, fuels, and specialty chemicals. Strain engineering focuses on manipulating and improving these hosts for new and enhanced functionalities including increased titers and better ...

Promoter substitutions are frequently used to regulate the expression of genes in a specific manner such as for their conditional expression or for their overexpression. Chromosomal integration of a regulatable promoter upstream of an open reading frame (ORF) by homologous recombi ...

Fully annotated genome sequences of many microorganisms are publicly available as a resource. However, in-depth analysis of these genomes using specialized tools is required to derive meaningful information. We describe here the utility of three powerful publicly available geno ...

Following the discovery of the structure of DNA in 1953, it became clear that scientists needed to be able to distinguish different DNA sequences. In 1975, Edward Southern published details of a new method for detecting DNA fragments based upon their specific sequence. An indication of the impor ...

Conformation-sensitive capillary electrophoresis (CSCE) is a rapid, high-throughput screening method that can be applied to any region of a genome for detection of sequence variants. Slab gel-based conformation-sensitive gel electrophoresis was first described by Ganguly et ...

Denaturing High-Performance Liquid Chromatography (dHPLC) is probably the most versatile and one of the most widely used mutation screening technologies. It benefits from a combination of relative technical simplicity and a very high sensitivity (mutation detection rate), app ...

Conformation sensitive gel electrophoresis (CSGE) is a rapid screening method for the detection of DNA sequence variation, specifically single-base changes or small insertions and deletions. It has been widely used for mutation screening in genetic disorders and for the detection of ...

Human papillomavirus (HPV) is an essential cofactor for cancer at many sites, including the genital tract, oral cavity, conjunctiva, and periungual region. The in situ detection of HPV allows us to determine the cellular targets of the virus. In situ-based coexpression analyses of HPV with put ...