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In this paper, we use a graded BDI agent model based on multi-context systems to specify an architecture for a Travel Assistant Agent that helps a tourist to choose holiday packages. We outline the theories of the different contexts and the bridge rules and illustrate the overall reasoning process of our model. [ABSTRACT FROM AUTHOR]

The extraction of frequent patterns often yields extremely voluminous results which are difficult to handle. Computing a concise representation or cover of the frequent pattern set is thus an interesting alternative investigated by various approaches. The work presented in this article fits in such a trend. We introduce the concept of essential pattern and propose a new cover based on this concept. Such a cover makes it possible to decide whether a pattern is frequent or not, to compute its frequency and, in contrast with related work, to infer its disjunction and negation frequencies. A levelwise algorithm with a pruning step which uses the maximal frequent patterns for computing the essential patterns is proposed. Experiments show that when the number of frequent patterns is very high (strongly correlated data), the defined cover is significantly more reduced than the cover considered until now as minimal: the frequent closed patterns. [ABSTRACT FROM AUTHOR]

Electroporation, originally developed as a method to introduce DNA into eukaryotic cells (1), has subsequently been extensively used for bacterial transformation (2,3). This procedure is an effective method for the transfer of DNA to a wide range of Gram-negative bacteria, such as Escherichia coli, and reports indicate that 109 electro-transformants per microgram of DNA can be achieved in this species (4,5). Electroporation is probably the most efficient and reliable method for the transformation of E. coli strains using plasmid DNA. [ABSTRACT FROM AUTHOR]

Green fluorescent protein (GFP) of the jellyfish Aqueorea victoria is a 238-amino-acid, 28-kDa protein that absorbs light with an excitation maximum of 395 nm and fluoresces with an emission maximum of 509 nm (1). GFP owes its unique spectral properties to its chromophore (2) that consists of a Ser65, Tyr66, and Gly67 tripeptide (3). Autocatalytic cyclization of this tripeptide, induced by oxidation of Tyr66, is a necessary posttranslational step for proper fluorescence (strong reducing agents reversibly convert GFP into a nonfluorescent form) (4). This can occur in the absence of any cofactors, making GFP an extremely useful tool for a wide range of applications in a variety of heterologous systems (5-7). GFP activity can be assayed both qualitatively and quantitatively using a variety of techniques, including simple plate counting, fluorescence and confocal microscopy, flow cytometry, and fluorometry. Transcriptional and translational fusions of GFP to a gene or protein of interest can be used as gene expression reporters and subcellular localization tags. GFP is a small protein (28 kDa) compared to other reporters (e.g., β-galactosidase is 465 kDa) and GFP fusions often retain the native protein function (8-10). This makes GFP useful as a generic tag for studying protein synthesis, translocation, and other protein-protein interactions. GFP is also widely used as a reporter in many genetic techniques, including transposon mutagenesis, promoter/enhancer traps, and one-component hybrid systems. GFP can be visualized using microscopy in both live and fixed cells, making it an excellent tool for studying dynamic changes in living cells. [ABSTRACT FROM AUTHOR]

The Gram-negative bacterium Escherichia coli enjoys widespread use in modern biology as both a model organism and a microbiological tool. One of the keys to its popularity lies in the functionality of the lac operon. This regulated E. coli operon has been manipulated and utilized in diverse and creative ways since it was first studied by Jacob and Monod in 1961 (1). Excellent in-depth reviews of the lac operon and its uses in classical genetics are provided in refs. 2 and 3. Its enduring use can be attributed to the development of a wide range of engineered mutants and plasmid constructs designed to take advantage of specific features of the operon, particularly its inducibility by β-galactosides. This chapter will focus on the use of the lac operon in E. coli protein expression, although it has also been utilized for protein expression in mammalian systems (4). The application of lacZ as a reporter gene is discussed in Chapter 30. [ABSTRACT FROM AUTHOR]

Escherichia coli is the most commonly used and best characterized organism for overexpressing foreign and nonforeign proteins. The use of E. coli confers several immediate advantages to the user: rapid and high-level expression as a result of the speed of cell growth to high density; low complexity and low cost of growth media; and the ability to target proteins to the desired subcellular location (1). In addition, a number of mutant host strains are available that can improve recombinant protein expression. For example, the use of host strains with mutations in cytoplasmic protease genes decreases protein degradation. Hosts that encode tRNAs for codons seldom found in E. coli can successfully express some otherwise difficult to express proteins that contain these rare codons (seeChapter 3). [ABSTRACT FROM AUTHOR]

In this chapter, we describe the use of plasmid vectors in transcription and translation systems in vitro to investigate aspects of the biology of the gene and the protein for which it codes. An in vitro, or cell-free, assay reproduces a relatively complex physiological process by mixing the essential purified components of the system under controlled conditions outside of the cell. Such systems allow the basic steps of transcription and translation to be studied individually, and the products obtained at each step to be altered in different ways according to the needs of the research. Thus, an in vitro system is convenient when it is necessary to modify a product, for example, by introducing mutations, labels, tags, or fusions. [ABSTRACT FROM AUTHOR]

Transposons are mobile genetic elements with the capacity to "jump" to new target DNA. Although first discovered in Zea mays by McClintock (1), they are present in DNA genomes of species from all kingdoms. Transposons fall into two major classes. Class I transposons are retroelements that transpose via an RNA intermediate that is synthesized by a reverse transcriptase (e.g., Alu elements in primates and Ty elements in yeast); class II transposons transpose directly from DNA to DNA (e.g., P elements in yeast and Tn elements in bacteria). This chapter describes the application of class II Tn elements from bacteria to mutagenize plasmid DNA by insertional mutagenesis. [ABSTRACT FROM AUTHOR]

DNA fragments cloned into plasmids are frequently greater than 500 base pairs in length and thus may be too long to sequence from a single primer-binding site in the vector. An efficient way to sequence such large DNA inserts is to generate a nested set of deletions in the target DNA, effectively moving the priming site closer to the sequence of interest. Similarly, nested deletions can be used to delineate a feature of interest (e.g., a replicon [1] or promoter) or to subclone a region of DNA devoid of restriction enzyme sites. [ABSTRACT FROM AUTHOR]

Site-directed mutagenesis has revolutionized the study of protein structure and function by enabling the controlled and systematic production of mutant proteins. Early methods of site-directed mutagenesis involved the use of a mutated oligonucleotide primer to prime synthesis of a target single-stranded DNA template. These approaches were very inefficient, yielding success rates of 1-5% (1). A dramatic improvement in the efficiency of generating mutations resulted from the use of single-stranded, uracil-containing DNA molecules isolated from ung− dut− Escherichia coli strains (seeChapter 3). Again, the mutation is introduced in a mutated oligonucleotide primer. Selection against the wild-type sequence parent DNA occurs on transformation into wild-type E. coli. Mutagenesis by this method was relatively efficient, with rates of 15-35%, but required a number of subcloning steps involving single-stranded M13 phage clones (2). It was only following the development of the polymerase chain reaction (PCR) that the two concepts were combined, dramatically improving the efficiency of the whole procedure. [ABSTRACT FROM AUTHOR]

Site-directed mutagenesis (SDM) is used to introduce a defined mutation into target DNA of known sequence to study, for example, gene expression or protein structure-function relationship. A number of polymerase chain reaction (PCR)-based mutagenesis methods have been developed (1). Among them, the "megaprimer" method is probably the simplest and most flexible (2-5). The megaprimer method utilizes two external oligonucleotide primers and one internal mutagenic primer in two rounds of PCR with a DNA template that contains the sequence to be mutated. The first round of PCR is carried out using one of the external primers and the mutagenic primer containing the desired mutation. This amplifies an intermediate PCR product that is purified and used as a "megaprimer" for the second round of PCR, along with the other external primer. The final PCR product is cloned into appropriate vectors and used in downstream applications. [ABSTRACT FROM AUTHOR]

The most widespread method used for DNA sequencing today is the Sanger dideoxy method that was first described in 1977 (1). This method takes advantage of the requirement for a free 3′ hydroxyl group to form the necessary phosphodiester bridge between two nucleotides during DNA polymerization. In short, DNA sequencing reactions using this method are performed on a DNA template using a specific primer, DNA polymerase, and a mixture of deoxynucleotidetriphosphates (dNTPs) and dideoxynucleotides (ddNTPs) as a substrate. When a dNTP is incorporated into a growing DNA strand, chain elongation proceeds normally. However, when a ddNTP is incorporated by the polymerase, the process of chain elongation is aborted because of the absence of the 3′ hydroxyl group on the nucleotide analog. The resulting DNA polymerization products are of varying lengths depending on when a ddNTP was incorporated into the growing strand. Theoretically, there should be a ddNTP incorporated at least once at each of the base positions for any particular DNA template. This pool of DNA fragments is then separated by size on high-resolution polyacrylamide gels. The resolution of these gels is so high that the difference in size resulting from a single nucleotide can be determined. [ABSTRACT FROM AUTHOR]

A recombinant DNA library typically represents part or all of an organism's genomic DNA or mRNA (represented as cDNA) cloned into vectors and stored as a collection of thousands of transformants. The construction of a complete library is only half the task; researchers then need to be able to identify the small number of clones bearing the DNA fragment of interest among the numerous transformants within the library. This process is called "screening a library" and it is the molecular equivalent of finding a needle in a haystack. Libraries may be screened by any one of several methods. [ABSTRACT FROM AUTHOR]

A key step in the construction of recombinant plasmids is the verification of the successful cloning of insert DNA into the vector. A number of commonly used plasmids facilitate phenotypic selection and/or screening methods for rapid identification of insert-containing clones. Additional screening methods are required to confirm the presence and/or orientation of the intended insert. A polymerase chain reaction (PCR) strategy may be sufficient to verify the presence and orientation of the intended insert depending on the availability of vector- and insert-specific primers (seeChapter 19). In situations where PCR-based analysis is undesirable or additional analysis is required, clear information about the clone can be derived from restriction analysis. [ABSTRACT FROM AUTHOR]

Construction of recombinant plasmid DNA is one of the cornerstones of molecular biology. The ability to clone DNA in a plasmid vector opens doors to downstream applications such as amplification of DNA, expression of desired genes, and construction of DNA libraries. Recombinant plasmids are generally constructed by first isolating the target DNA and linearizing the plasmid vector of choice (seeChapter 2). The insert DNA is subsequently ligated to the vector DNA (seeChapters 15 and 17) and the ligation mixture transformed into an appropriate Escherichia coli host (seeChapters 3-5) (1,2). E. coli transformed with recombinant plasmid or self-ligated vector will both grow on appropriate selective medium; therefore, screening is almost always necessary to select colonies containing the recombinant plasmid and, in some cases, with the correct orientation of insert. [ABSTRACT FROM AUTHOR]

Lambda (λ) bacteriophages are viruses that specifically infect bacteria. The genome of λ-phage is a double-stranded DNA molecule approx 50 kb in length (1). In bacterial cells, λ-phage employs one of two pathways of replication: lytic or lysogenic. Commonly, λ-phage vectors replicate via the lytic pathway. During lytic growth, the viral DNA is replicated manifold, a large number of phage gene products are synthesized, and progeny phage particles are assembled. The cell is eventually lysed, releasing its many new infectious virus particles; at this time, plaques will form on an infected bacterial lawn. Its high infectivity and clone-style propagation are the inherent basis for the use of λ-phage as a vector. Moreover, the central third of the viral genome is not essential for lytic growth and, thus, can be replaced by a variety of foreign gene segments. λ-Vectors usually contain multiple cloning sites facilitating the cloning of foreign DNA. In addition, λ-phage vectors have the following advantageous features: high cloning efficiency, a relatively large insert-size capacity, and suitability for screening using nucleic acid probes. In terms of gene screening, genomic DNA libraries are often screened by hybridization using a radioactive nucleic acid probe. [ABSTRACT FROM AUTHOR]

Since it was described in 1988 (1), the polymerase chain reaction (PCR) has been a valuable tool for molecular biologists. PCR allows researchers to produce a large quantity of a desired DNA fragment while requiring only a small amount of template. Prior to PCR, isolation of DNA fragments was typically performed by cleavage of the DNA with restriction endonuclease enzymes. The relative abundance or scarcity of appropriate restriction sites within the region of interest greatly affected the ability of researchers to obtain specific DNA fragments. PCR gives researchers the ability to establish the terminal sequences as well as the size of the amplified fragment and, in this way, provides freedom from the problems associated with location and abundance of restriction enzyme recognition sites. [ABSTRACT FROM AUTHOR]

A fundamental step in molecular biology is the cloning of a DNA fragment insert into a plasmid vector. This allows the cloned fragment to be replicated upon transformation of the recombinant molecule into a bacterial cell (seeChapters 4 and 5) so that the DNA of interest can be investigated further. Cloning is an essential part of many experiments, including library generation (seeChapter 18) and expression studies (seeChapter 29). [ABSTRACT FROM AUTHOR]

Manipulation and analysis of DNA sequences is often a complex task involving many steps, each of which must be carefully planned and executed. To facilitate this process, the number of steps should be minimized and each step analyzed to ensure that it has been completed successfully. Often, this analysis involves restriction-enzyme digestion of DNA constructs followed by gel electrophoresis to examine the results. It is also important to be able to easily track the DNA manipulations. This means that each step must be documented, illustrated, and stored as the project proceeds. It is also desirable to maintain an overview of the cloning strategy and track changes as they are made. Graphical representations of DNA constructs make the process simple to follow and less prone to errors. [ABSTRACT FROM AUTHOR]

Single-stranded DNA (ssDNA) is the optimal template for most polymerase-based molecular-biology applications, including DNA sequencing and site-directed mutagenesis. Phagemids are chimeric vectors, derived from the ssDNA bacteriophages M13, fd, or f1, that normally replicate as plasmids in bacterial hosts (1) (seeChapter 2). Phagemids typically lack the genes required for packaging into virions and, thus, "helper" bacteriophage are often required to package phagemids as ssDNA into viral particles, which are subsequently released into the culture medium (2). ssDNA isolated from these virions is suitable for use as a template in a variety of procedures with no further manipulation, as denaturation is not required prior to its use as a template. [ABSTRACT FROM AUTHOR]

Cosmid and bacterial artificial chromosome (BAC) systems have been developed for the cloning of large DNA inserts averaging 40 kb and 130 kb (range: 90-300 kb), respectively. The resulting clones are more stable than yeast artificial chromosomes (YACs) and rarely chimeric, which makes them excellent tools for the generation of contiguous physical maps. The use of such large-insert clones considerably increases the rate of complex genome mapping and sequencing. Numerous standard protocols have been developed to isolate supercoiled plasmids, but of these, the alkaline lysis protocol remains the most suitable approach to isolate large-insert clones such as cosmids and BACs. [ABSTRACT FROM AUTHOR]

Plasmid extraction is typically performed to produce template DNA for a desired molecular biological reaction, or set of reactions, such as restriction endonuclease digestion (seeChapter 20), DNA sequencing (seeChapter 22), in vitro mutagenesis (seeChapters 23-26), transformation (seeChapters 4 and 5), transfection, or probe generation. The principal methodologies of extraction, involving either alkaline lysis or boiling are well established (1) and are described in detail in Chapters 8 and 9. These methods are relatively crude but are both inexpensive and rapid, and they yield DNA that is suitable for many molecular biology protocols. DNA of higher purity can be prepared using silica resin-based anion exchange as described in Chapter 10 or by separation on cesium chloride gradients (for details, seeref. 1, Chapter 1, Protocol 10). Plasmid minipreps are a ubiquitous part of molecular biology. They are simple and easy to perform but can be very tedious when large numbers of samples are involved. In recent years, endeavors such as the human genome project have led to the development of methods for the manageable high-throughput extraction of plasmids (2-15). [ABSTRACT FROM AUTHOR]

The isolation of plasmid DNA from bacteria is a crucial technique in molecular biology and is an essential step in many procedures such as cloning, DNA sequencing, transfection, and gene therapy. These manipulations require the isolation of high-purity plasmid DNA. Commercial anion-exchange columns, even though widely used, are relatively costly. Less expensive methods that use silica oxide as the DNA-binding matrix have the disadvantage that bacterial lipopolysaccharide (LPS), or endotoxin, is copurified and can inhibit downstream applications. In particular, the LPS content of DNA used for transfection heavily influences the efficiency (1-3). Furthermore, these methods usually require the use of hazardous chemicals (e.g., guanidine hydrochloride), which act as chaotropic substances, changing the structure of water to facilitate the binding of the DNA to silica oxide (4). [ABSTRACT FROM AUTHOR]

The boiling lysis procedure (1) is quick to perform and, therefore, especially suitable for screening large numbers of small-volume Escherichia coli cultures. It is described with different adaptations in a variety of protocol books (2,3). The quality of the isolated plasmid DNA is lower than that from an alkaline lysis miniprep, but it is sufficient for restriction analysis. [ABSTRACT FROM AUTHOR]

Purification of plasmid DNA from Escherichia coli using alkaline lysis (1,2) is based on the differential denaturation of chromosomal and plasmid DNA in order to separate the two. Bacteria are lysed with a solution containing sodium dodecyl sulfate (SDS) and sodium hydroxide. During this step, chromosomal as well as plasmid DNA are denatured. Subsequent neutralization with potassium acetate allows only the covalently closed plasmid DNA to reanneal and to stay solubilized. Most of the chromosomal DNA and proteins precipitate in a complex formed with potassium and SDS, which is removed by centrifugation. The plasmid DNA is concentrated from the supernatant by ethanol precipitation. Using this procedure, 2-5 μg of DNA can be obtained from a 1.5-mL culture of E. coli containing a pBR322-derived plasmid, and three- to five-fold higher yields can be expected from pUC-derived plasmids (3). [ABSTRACT FROM AUTHOR]

Cosmids are cloning vectors that were developed to enable large fragments of DNA to be cloned and maintained (1-3). Cosmid vectors allow the cloning of fragments up to 45 kilobases (kb) and are commonly used in genomic library construction. The distinguishing feature of a cosmid is the presence of bacteriophage λ cos sites, which enable the vector, and cloned genomic DNA, to be packaged into bacteriophage heads (1,4). Cosmids also contain several other components, including a drug-resistance marker that is necessary for the selection of cosmid-containing bacteria, a plasmid origin of replication, usually ColE1, which regulates cosmid replication and copy number within the Escherichia coli host (1,5), and restriction sites that facilitate cloning of the desired DNA fragments and insert verification by restriction digestion (see Fig. 1). [ABSTRACT FROM AUTHOR]

Bacterial conjugation is defined as contact-dependent transmission of genetic information from a donor bacterium to a recipient cell (1). Transfer of DNA by conjugation is often termed lateral or horizontal gene transfer, as opposed to vertical transfer by which genetic information is transferred from mother to daughter cells. When genetic information is transferred by conjugation from the donor strain to the recipient strain, this population of recipient bacteria are called transconjugants. The genetic information is usually transferred to the recipient bacterium on a plasmid; however, conjugative transposons are also known. The mechanism and proteins involved in conjugative DNA transfer vary depending on the type of plasmid or transposon present in the donor strain. For example, RP4 and F (fertility) plasmids isolated from Escherichia coli are self-transmissible plasmids and thus contain genes that encode for all of the necessary proteins involved in the mobilization and transfer of the plasmid from one bacterium to another (2). Other plasmids that are conjugative, but non-self-transmissible, such as RSF1010 isolated from E. coli, can only be mobilized when the necessary functions are supplied in trans (3). The cointegration of a conjugative and nonconjugative circular plasmid can result in the transfer of both plasmids into a recipient strain. For example, in a high-frequency recombinant (Hfr) E. coli K-12 strain, where the F plasmid has integrated into the bacterial chromosome, the whole chromosome can be conjugated to a recipient strain (4,5). Finally, conjugative transposons can be transferred from one bacterium to another. [ABSTRACT FROM AUTHOR]

Transformation is defined as the transfer of genetic information into a recipient bacterium using naked DNA, without any requirement for contact with a donor bacterium. The ability to transform or accept exogenous DNA is generally referred to as competence, although the term has been so widely used in different systems that it is difficult to generate an all-inclusive definition for competence. Natural competence occurs in a defined subset of bacterial species that have the capacity to take up linear, and sometimes circular, DNA, usually dependent on a specific uptake system. As natural competence is restricted to a subset of bacteria, methods for the chemical induction of a competent state in otherwise nontransformable bacteria are an important tool in bacterial genetics. For these species, competence refers to the ability to take up and propagate plasmid DNA, usually with no sequence specificity for uptake. [ABSTRACT FROM AUTHOR]

To successfully perform molecular genetic techniques it is essential to have a full understanding of the properties of the various Escherichia coli host strains commonly used for the propagation and manipulation of recombinant DNA. E. coli is an enteric rod-shaped Gram-negative bacterium with a circular genome of 4.6 Mb (1). It was originally chosen as a model system because of its ability to grow on chemically defined media and its rapid growth rate. In rich media, during the exponential phase of its growth, E. coli doubles every 20-30 min; thus, during an overnight incubation period, a single selected organism will double enough times to yield a colony on an agar plate, or 1-2 billion cells per milliliter of liquid media. The ease of its trans-formability and genetic manipulation has subsequently solidified the role of E. coli as the host of choice for the propagation, manipulation, and characterization of recombinant DNA. In the past 60 yr E. coli has been the subject of intensive research and more is now known about these bacilli than any other organisms on earth. [ABSTRACT FROM AUTHOR]

Since the construction of the first generation of general cloning vectors in the early 1970s, the number of plasmids created has increased to an almost countless number. Thus, a critical decision facing today's investigator is that of which plasmid to use in a particular project? Despite the bewildering choice of commercial and other available vectors, the choice of which cloning vector to use can be decided by applying a small number of criteria: insert size, copy number, incompatibility, selectable marker, cloning sites, and specialized vector functions. Several of these criteria are dependent on each other. This chapter discusses these criteria in the context of choosing a plasmid for use as a cloning vector and Table 1 displays the features of some commonly used cloning vectors. [ABSTRACT FROM AUTHOR]

In 1952, Joshua Lederberg coined the term plasmid to describe any bacterial genetic element that exists in an extrachromosomal state for at least part of its replication cycle (1). As this description included bacterial viruses, the definition of what constitutes a plasmid was subsequently refined to describe exclusively or predominantly extrachromosomal genetic elements that replicate autonomously. Plasmids are now known to be present in most species of Eubacteria that have been examined, as well as in Archaea and lower Eukarya (2). [ABSTRACT FROM AUTHOR]

Near-infrared images of the Serpens molecular cloud core have been carried out at UKIRT (Mauna Kea Observatory) using the infrared array camera, IRCAM. A large-scale diffuse nebulosity extending over the central part of the core is observed. Over 100 K-sources are detected in the 30 arc min2 cloud core. Some of them are PMS objects which were previously unknown. For the first time, a near-infrared counter-part of the far-infrared source Serpens FIRS1 has been detected. The 2.2 µm source appears as a point like object at the apex of an extended knotty, jet-like nebulosity oriented towards the northwest. In addition, a group of 11 stellar objects is seen in the position of the IR/radio source SVS4. These objects are embedded in a very faint nebulosity and form one of the densest clustering of young stars found in dark clouds. [ABSTRACT FROM AUTHOR]

A common step in cloning experiments is the purification of DNA fragments prior to ligation. Often, both the insert and vector DNA fragments will be derived from restriction endonuclease digests and, thus, will be mixed with enzymes, salts, and possibly other DNA fragments that may inhibit the ensuing ligation reaction. A simple solution to this problem is to purify the DNA fragments of interest. This often involves agarose gel electrophoresis to separate mixtures of DNA fragments, followed by extraction of the DNA fragment of interest from the gel. The importance of this step is obvious from the fact that every vendor of molecular-biology products produces a gel extraction kit. Most of these kits utilize a chaotropic agent, such as sodium iodide, to destabilize the agarose gel. The DNA is subsequently bound to a substrate (e.g., an anionic resin) and washed to remove impurities prior to elution of the DNA from the substrate. Other methods include hot phenol extraction of the DNA from the gel. The use of phenol is best avoided if possible because of safety and waste disposal concerns. Presented here are two alternative techniques that avoid these concerns, do not require the purchase of a specialized kit, and are simple to perform. [ABSTRACT FROM AUTHOR]