TNBGGA: The No Bullshit Guide to Genetic Analysis

The same types of genetic analysis we performed on yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 and Drosophilaplugin-autotooltip__default plugin-autotooltip_bigDrosophila melanogaster: a fruit fly species used in genetics research. in earlier chapters, such as tests for dominanceplugin-autotooltip__default plugin-autotooltip_bigDominant: used to describe an allele, usually in comparison to wildtype. Dominant alleles will express their phenotype when combined with a wildtype allele. or for complementationplugin-autotooltip__default plugin-autotooltip_bigComplementation: a concept where an additional allele of a gene (usually a wildtype allele) can provide normal function to an organism with a recessive loss of function mutation in that gene. The concept of complementation underlies the complementation test., can in principle be applied to bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. as well. However, these tests rely on organisms being diploidplugin-autotooltip__default plugin-autotooltip_bigDiploid: a term that describes a cell or organism that has two copies of similar genetic information, usually obtaining one copy from a male parent and the other copy from a female parent., and bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. are haploidplugin-autotooltip__default plugin-autotooltip_bigHaploid: a term that describes a cell or organism that has only one copy of genetic information. Haploid cells typically arise from meiosis (or mitosis of a haploid mother cell).. To do these tests, we need a way to make the bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. diploidplugin-autotooltip__default plugin-autotooltip_bigDiploid: a term that describes a cell or organism that has two copies of similar genetic information, usually obtaining one copy from a male parent and the other copy from a female parent., or at least partially diploidplugin-autotooltip__default plugin-autotooltip_bigDiploid: a term that describes a cell or organism that has two copies of similar genetic information, usually obtaining one copy from a male parent and the other copy from a female parent.. One way to do this is to use an extrachromosomal genetic element, or episomeplugin-autotooltip__default plugin-autotooltip_bigEpisome: a piece of DNA that exists separately from chromosomes and that can replicate and segregate during cell division. While all episomes are natural in origin, they are commonly exploited by researchers for genetics research., called an F plasmidplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids (Fig. 1), a circular double-stranded DNAplugin-autotooltip__default plugin-autotooltip_bigDouble-stranded DNA (dsDNA): DNA that consists of two complementary strands of ssDNA paired together via hydrogen bonds between the nitrogenous bases G, A, T, and C. (dsDNAplugin-autotooltip__default plugin-autotooltip_bigDouble-stranded DNA (dsDNA): DNA that consists of two complementary strands of ssDNA paired together via hydrogen bonds between the nitrogenous bases G, A, T, and C.) molecule that can be transferred from one cell to another. The “F” stands for fertility, and F plasmidplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids transfer is one version of “sex ” in E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs)..

There are some special terms to describe the state of F plasmidsplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids in a cell. F^– refers to a strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and without any form of an F plasmidplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids, whereas F⁺ refers to a strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and with an F plasmidplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids. We can also call these strains recipient and donor strains, respectively. An F plasmidplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids (hereafter referred to as simply F) is pretty efficient at transferring itself from an F⁺ cell to an F^- cell. After culturing F⁺ and F^- cells together about 10% of the F^- cells will become F⁺. F⁺ cells form a structure called a pilus, which is a tunnel through which F can transfer into the F^- cell through a rolling circle mechanism (Fig. 2). The F plasmidplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. moves to the recipient cell linearly instead of as a circle.

The property that makes F useful for genetic manipulation is that it will integrate into E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. at low frequency. This can happen at multiple locations on the chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. and occurs because F contains DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. called insertion sequences that are also present at multiple locations on the E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins.. Crossing overplugin-autotooltip__default plugin-autotooltip_bigCrossing over: an event where non-sister chromatids exchange material with each other during meiosis I. (recombinationplugin-autotooltip__default plugin-autotooltip_bigRecombination: Recombination can have slightly different meanings depending on context:

* In the context of genetic crosses (usually a dihybrid cross or a test cross), recombination refers to the phenomena where the phenotype of the F2 offspring is different than either parent (P generation). $lox$) between insertion sequences on F and on the chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. results in integration.

An E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and with F integrated into the chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. will give efficient transfer of some chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. markersplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). to an F^- cell. It does so by excising itself from the chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. to initiate transfer. Such a strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and is called Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids (for high frequency recombinationplugin-autotooltip__default plugin-autotooltip_bigRecombination: Recombination can have slightly different meanings depending on context:

* In the context of genetic crosses (usually a dihybrid cross or a test cross), recombination refers to the phenomena where the phenotype of the F2 offspring is different than either parent (P generation). $lox$). Note that a markerplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). is simply a locusplugin-autotooltip__default plugin-autotooltip_bigLocus (plural form: loci): a physical location of a gene; often used as a synonym for a gene.¹⁾ that has allelesplugin-autotooltip__default plugin-autotooltip_bigAllele: a version of a gene. Alleles of a gene are different if they have differences in their DNA sequence. that confer an easily observable phenotypeplugin-autotooltip__default plugin-autotooltip_bigPhenotype: an observable feature or property of an organism.. There are different Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids strains, and each strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and efficiently transfers different sets of chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. markersplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). depending on where the F was integrated into the E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins.. Since F plasmidplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. transfers linearly (Fig. 2), markersplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). closer to the F insertion site transfer earlier, and markersplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). farther away from the F insertion site transfer later. You can measure distances using time of transfer for markersplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). during Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids transfer (using the unit “minutes”), as opposed to frequency of recombinationplugin-autotooltip__default plugin-autotooltip_bigRecombination: Recombination can have slightly different meanings depending on context:

* In the context of genetic crosses (usually a dihybrid cross or a test cross), recombination refers to the phenomena where the phenotype of the F2 offspring is different than either parent (P generation). $lox$ in regular genetic mappingplugin-autotooltip__default plugin-autotooltip_bigGenetic mapping: a term describing a variety of different experimental approaches used to determine the physical locations of genes on chromosomes. in other organisms. Theoretically it would take about 100 minutes to transfer the entire E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. from an Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and to F^-, although an Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids would not usually be able to transfer the whole chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins.; this just gives you a rough idea of how fast the Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids transfer rate is. Thus, you can say that the size of the E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). genomeplugin-autotooltip__default plugin-autotooltip_bigGenome: a dataset that contains all DNA information of an organism. Most of the time, this also includes annotation and curation of that information, e.g., the names, locations, and functions of genes within the genome. As an adjective (“genomic”), this usually is used in the context of is 100 minutes when measured by Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids transfer. As with recombinationplugin-autotooltip__default plugin-autotooltip_bigRecombination: Recombination can have slightly different meanings depending on context:

* In the context of genetic crosses (usually a dihybrid cross or a test cross), recombination refers to the phenomena where the phenotype of the F2 offspring is different than either parent (P generation). $lox$ mappingplugin-autotooltip__default plugin-autotooltip_bigGenetic mapping: a term describing a variety of different experimental approaches used to determine the physical locations of genes on chromosomes. we studied in Chap. 05, you can add map distances of markersplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). together (using minutes as your unit of measure) to generate a map of the E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). genomeplugin-autotooltip__default plugin-autotooltip_bigGenome: a dataset that contains all DNA information of an organism. Most of the time, this also includes annotation and curation of that information, e.g., the names, locations, and functions of genes within the genome. As an adjective (“genomic”), this usually is used in the context of.

How does an Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and transfer chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. markersplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). to a recipient strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and? Consider an F⁺ integrating in the E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. via homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species. to make an Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids (Fig. 4). Homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species. in E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). uses different molecular mechanisms than crossing overplugin-autotooltip__default plugin-autotooltip_bigCrossing over: an event where non-sister chromatids exchange material with each other during meiosis I. during eukaryoticplugin-autotooltip__default plugin-autotooltip_bigeukaryote: organism whose cells have membrane bound organelles, including the nucleus. meiosisplugin-autotooltip__default plugin-autotooltip_bigMeiosis: a process involving two sequential cell divisions that usually produces four gametes (reproductive cells such as sperm or eggs)., but the overall process in terms of lining up highly similar DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. and following the “chiasmaplugin-autotooltip__default plugin-autotooltip_bigChiasma (plural: chiasmata): structures shaped like the Greek letter χ (usually Romanized as chi but pronounced “kai”) that form between non-sister chromatids during meiosis I. Chiasmata is the physical manifestation of crossing over. “Crossover” can be used as a synonym for chiasma.”²⁾ to determine the recombinantplugin-autotooltip__default plugin-autotooltip_bigRecombinant: (adj.) Describing something that has undergone recombination, e.g., recombinant DNA or recombinant offspring. “Non-parental” is a synonym when referring to organisms. (n.) Something that has undergone recombination, e.g., “This fly is a recombinant.” DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. structure is similar.

This process can be reversed to go back to the F⁺ state (Fig. 5). The chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. with the integrated F loops back on itself so that the integration sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. lineplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and up with each other.

Homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species. can sometimes occur at a different position to excise an F plasmidplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids that carries a part of the E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins.. In the example in Fig. 6, this is the BC segment of the E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins.. This form of F is called an F' (pronounced “F prime”).

F' plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. are usually isolated by selectionplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a for early transfer of a markerplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). that is transferred late in the Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids. In the example above the F' could have been isolated from a population of Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. by selectingplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a for early transfer of either B or C. In our example, the Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids in Fig. 5 would transfer B efficiently (earlier) but C less efficiently (later). By comparison, the F' in Fig 6 will transfer both B and C efficiently. Also, the Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids (Fig. 5) might transfer markerplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). D given enough time, but F' (Fig. 6) would never be able to transfer markerplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). D.

F' plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. can be used to perform genetic tests of function (i.e., complementationplugin-autotooltip__default plugin-autotooltip_bigComplementation: a concept where an additional allele of a gene (usually a wildtype allele) can provide normal function to an organism with a recessive loss of function mutation in that gene. The concept of complementation underlies the complementation test. tests) because a cell containing a F' will be diploidplugin-autotooltip__default plugin-autotooltip_bigDiploid: a term that describes a cell or organism that has two copies of similar genetic information, usually obtaining one copy from a male parent and the other copy from a female parent. for the region of the chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. carried on F. This type of partial diploidplugin-autotooltip__default plugin-autotooltip_bigDiploid: a term that describes a cell or organism that has two copies of similar genetic information, usually obtaining one copy from a male parent and the other copy from a female parent. is known as a merodiploidplugin-autotooltip__default plugin-autotooltip_bigMerodiploid: used to describe an organism that is naturally haploid but is partially diploid for a portion of its genome due to the presence of an episome that contains a chromosomal fragment.. For example, if we isolated a new $lacZ$ mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. we could use an F' that carries $lacZ^+$ to determine whether this mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. is dominantplugin-autotooltip__default plugin-autotooltip_bigDominant: used to describe an allele, usually in comparison to wildtype. Dominant alleles will express their phenotype when combined with a wildtype allele. or recessiveplugin-autotooltip__default plugin-autotooltip_bigRecessive: used to describe an allele, usually in comparison to wildtype. Recessive alleles do not exhibit their phenotype when combined with a wildtype allele..

With this tool, we are able to perform many of the same genetic analyses on mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. in E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). as we were able to with yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 and Drosophilaplugin-autotooltip__default plugin-autotooltip_bigDrosophila melanogaster: a fruit fly species used in genetics research..

F and F' are one type of many different kinds of bacterialplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors., most of which are also transmissible from one cell to another. R factorsplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning. (also called R plasmidsplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning.) are a different kind of plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. that was discovered in Japan in the early 1950s. They came from hospital patients that were infected with bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. that were resistant to several different kinds of antibioticsplugin-autotooltip__default plugin-autotooltip_bigAntibiotics: naturally produced compounds, usually found in molds and fungi but are typically commercially available, that either kill bacteria or prevent their replication. In genetics research, they are often used to select for the presence of an episome that carries an antibiotic resistance. This was surprising since different antibioticsplugin-autotooltip__default plugin-autotooltip_bigAntibiotics: naturally produced compounds, usually found in molds and fungi but are typically commercially available, that either kill bacteria or prevent their replication. In genetics research, they are often used to select for the presence of an episome that carries an antibiotic resistance work by very different mechanisms. For example, resistance to ampicillinplugin-autotooltip__default plugin-autotooltip_bigAmpicillin: a common type of antibiotic used in genetics research., kanamycin, tetracyclineplugin-autotooltip__default plugin-autotooltip_bigTetracycline: a common type of antibiotic used in genetics research., and sulfonamide could be conferred to antibioticplugin-autotooltip__default plugin-autotooltip_bigAntibiotics: naturally produced compounds, usually found in molds and fungi but are typically commercially available, that either kill bacteria or prevent their replication. In genetics research, they are often used to select for the presence of an episome that carries an antibiotic resistance-sensitive bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. on transfer of an R factorplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning. (Fig. 7). Whereas F plasmidsplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids are usually present at one copy per cell, R plasmidsplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning. (at least modified versions) can exist at much higher copy numbers - up to 500-1000 copies per cell. R plasmidsplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning. have been engineered by scientists to use as cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of vectorsplugin-autotooltip__default plugin-autotooltip_bigVector: in molecular genetics, a vector is a genetic element (usually an episome of sort such as a plasmid but can be something like a virus) that is used to clone and/or transfer genes into and out of cells. – tools that help with molecular cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of³⁾.

Geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of refers to the ability to experimentally replicate a piece of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. containing a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (or a portion of a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), or really any piece of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth.) to produce large quantities of that DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth.. We saw in Chap. 07 that large quantities of any segment of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. can be produced using PCRplugin-autotooltip__default plugin-autotooltip_bigPolymerase chain reaction (PCR): An experimental technique invented by Kary Mullis used to exponentially amplify DNA in vitro. PCR made obtaining large quantities of DNA for analysis much faster and easier than using traditional cloning methods., but in some cases you need more DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. than can be produced by PCRplugin-autotooltip__default plugin-autotooltip_bigPolymerase chain reaction (PCR): An experimental technique invented by Kary Mullis used to exponentially amplify DNA in vitro. PCR made obtaining large quantities of DNA for analysis much faster and easier than using traditional cloning methods., and in other cases you might need to cloneplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. for which you don't have any prior knowledge of its sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids., which is a requirement for PCRplugin-autotooltip__default plugin-autotooltip_bigPolymerase chain reaction (PCR): An experimental technique invented by Kary Mullis used to exponentially amplify DNA in vitro. PCR made obtaining large quantities of DNA for analysis much faster and easier than using traditional cloning methods..

Geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of typically utilizes engineered and stripped-down versions of R factorsplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning. (Fig. 8). They usually carry a drug resistance geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (so that you can selectplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a for the presence of the plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors.) and an origin of replicationplugin-autotooltip__default plugin-autotooltip_bigDNA replication: usually the process of starting with a dsDNA molecule and ending with two identical copies of that dsDNA molecule. In most cases, “replication” implies DNA replication.⁴⁾ (so that the plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. can replicate in E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs).). R factorsplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning. in a test tube can be transformedplugin-autotooltip__default plugin-autotooltip_bigTransformation: in microbiology (including for yeasts), transformation is the alteration of phenotype due to uptake of external DNA into a cell. Not to be confused with “transformation” in the context of cell biology, where the definition is the alteration of a normal cell to a cancerous cell., or introduced, into E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). cells that have been treated with CaCl₂. Cells treated in such a way are called competent cells (i.e., they are “competent” to take up foreign DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth.). In Chapter 06, we briefly discussed the “transforming principle” discovered by Griffith - the term “transformationplugin-autotooltip__default plugin-autotooltip_bigTransformation: in microbiology (including for yeasts), transformation is the alteration of phenotype due to uptake of external DNA into a cell. Not to be confused with “transformation” in the context of cell biology, where the definition is the alteration of a normal cell to a cancerous cell.” used here comes from Griffith's usage of the word. Note that in cancer biology “transformationplugin-autotooltip__default plugin-autotooltip_bigTransformation: in microbiology (including for yeasts), transformation is the alteration of phenotype due to uptake of external DNA into a cell. Not to be confused with “transformation” in the context of cell biology, where the definition is the alteration of a normal cell to a cancerous cell.” means something different and it's important not to confuse the usage of this word in different contexts.

Molecular cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of involves the use of enzymesplugin-autotooltip__default plugin-autotooltip_bigEnzyme: a macromolecule, usually a protein (but sometimes an RNA), that functions as a catalyst of some kind of biochemical reaction. in vitroplugin-autotooltip__default plugin-autotooltip_bigIn vitro: taking place outside of a living organism, usually in a test tube or Petri dish. to make plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. carrying pieces of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. that you wish to be replicated. One of these important enzymesplugin-autotooltip__default plugin-autotooltip_bigEnzyme: a macromolecule, usually a protein (but sometimes an RNA), that functions as a catalyst of some kind of biochemical reaction. can cleave DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. at specific sites. These enzymesplugin-autotooltip__default plugin-autotooltip_bigEnzyme: a macromolecule, usually a protein (but sometimes an RNA), that functions as a catalyst of some kind of biochemical reaction. are known as restriction enzymesplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences.. They were discovered when it was found that bacteriophageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. λ (Greek letter lambda) infected different E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). strains at different efficiencies. Bacteriophagesplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. are viruses that infect and kill bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. when the phageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. reproduces. When a lawn of bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. covering the surface of a Petri dishplugin-autotooltip__default plugin-autotooltip_bigPetri dish: a round dish, usually 5-10 cm in diameter, that can contain growth media to grow cells in vitro. are killed by phageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria., a clear spot on the dish called a plaqueplugin-autotooltip__default plugin-autotooltip_bigPlaque: in bacteriology and genetics, a plaque forms in a bacteriophage infection experiment. Bacteria are grown as a lawn (i.e., they evenly cover the entire surface of a Petri dish), then injected with bacteriophage. The phage will kill and lyse bacterial cells, leaving a clear spot on the lawn. This clear spot is called a plaque. Plaques contain is formed (Fig. 9.9).

Plaquesplugin-autotooltip__default plugin-autotooltip_bigPlaque: in bacteriology and genetics, a plaque forms in a bacteriophage infection experiment. Bacteria are grown as a lawn (i.e., they evenly cover the entire surface of a Petri dish), then injected with bacteriophage. The phage will kill and lyse bacterial cells, leaving a clear spot on the lawn. This clear spot is called a plaque. Plaques contain can be counted to measure how efficient a phageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. is at infecting bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably.. Phageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. grown using E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and C are much less efficient at infecting E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and K (Table 2). But phageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. grown using strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and C can infect strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and C efficiently, just as phageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. grown using strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and K can infect strains C or K efficiently. This phenomenon is known as host restrictionplugin-autotooltip__default plugin-autotooltip_bigHost restriction: a phenotype in bacteria wherein a bacterial strain does not permit a bacteriophage to replicate., and it behaves like a genetic trait that reverts at a high frequency.

The explanation is that E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and K makes a restriction enzymeplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. that cleaves DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth., including λ DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth.. The K strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and doesn’t destroy its own chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. because it also makes a DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. methylase that enzymatically modifies nucleotideplugin-autotooltip__default plugin-autotooltip_bigNucleotide: molecules that are polymerized to form nucleic acids (DNA or RNA). Includes dNTPs and NTPs. bases at the cleavage site and thus prevents the restriction enzymeplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. from working. By rare chance, a small amount of λ phageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. can grow on strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and K because they escaped cleavage long enough to be modified. Subsequently, when λ phageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. are produced in strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and K, the phageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. is also modified and thus protected from cleavage. λ phageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. produced in strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and C is not modified, and therefore when these phagesplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. are used to infect strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and C, the infection efficiency is much lower as its DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. is cleaved by the restriction enzymeplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences..

The E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) for restriction enzymesplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. usually come in pairs organized in an operonplugin-autotooltip__default plugin-autotooltip_bigOperon: two or more genes that are transcribed together on an mRNA from a single promoter. Most commonly seen in bacteria; very rare in eukaryotes. (more on operonsplugin-autotooltip__default plugin-autotooltip_bigOperon: two or more genes that are transcribed together on an mRNA from a single promoter. Most commonly seen in bacteria; very rare in eukaryotes. in Chap. 10), with the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) for the restriction enzymeplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. ($R$) residing next to the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) for the DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. methylase that modifies the same sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. ($M$) (Fig. 9).

Mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. that have a mutated version of the restriction enzymeplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. but a wildtypeplugin-autotooltip__default plugin-autotooltip_bigWildtype: a reference strain of an organism that scientists operationally define as “normal” to which mutants are compared. Not to be confused with wild organisms. version of the modifying enzymeplugin-autotooltip__default plugin-autotooltip_bigEnzyme: a macromolecule, usually a protein (but sometimes an RNA), that functions as a catalyst of some kind of biochemical reaction. ($R^- M^+$) are useful for studying phageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. because they do not show host restrictionplugin-autotooltip__default plugin-autotooltip_bigHost restriction: a phenotype in bacteria wherein a bacterial strain does not permit a bacteriophage to replicate., but phageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. grown on these strains are resistant to host restrictionplugin-autotooltip__default plugin-autotooltip_bigHost restriction: a phenotype in bacteria wherein a bacterial strain does not permit a bacteriophage to replicate.. We don't generally consider phageplugin-autotooltip__default plugin-autotooltip_bigBacteriophage: viruses that infect bacteria. genetics in this course further, even though it's pretty interesting.

A large number of restriction enzymesplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. have been isolated from different bacterialplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. species and are commercially available. Most of the enzymesplugin-autotooltip__default plugin-autotooltip_bigEnzyme: a macromolecule, usually a protein (but sometimes an RNA), that functions as a catalyst of some kind of biochemical reaction. recognize palindromic DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. of 4 or 6 base pairsplugin-autotooltip__default plugin-autotooltip_bigBase pair: a term used to describe how nitrogenous bases (G, A, T/U, and C) in nucleic acids interact with each other via hydrogen bonds to form double-stranded molecules (including dsDNA, dsRNA, and DNA/RNA hybrids). G always pairs with C, and T/U always pairs with A.. A palindrome (when talked about in an everyday sense) is a sentence that reads exactly the same when read forward or backwards (e.g., “A man, a plan, a canal, Panama”). A palindromic sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. is a DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. that is the same on one strand and its reverse complementplugin-autotooltip__default plugin-autotooltip_bigComplement: used to describe the relationship between two recessive mutants. If a diploid created by mating the two mutants has a wildtype phenotype, we say the two mutants complement each other. strand. The 6 bp palindrome below is recognized by a restriction enzymeplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. called EcoRI (pronounced “eh-co-ar one”):

5’-GAATTC-3’
   ||||||
3’-CTTAAG-5’

Restriction enzymesplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. can be used to cut chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. into fragments. These fragments can be ligated (joined) into plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. that has also been cut with the same restriction enzymesplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences.. This procedure takes advantage of the fact that in most cases the DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. ends that remain after cleavage with a restriction enzymeplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. (called sticky ends) will base pair with other ends cut with the same enzymeplugin-autotooltip__default plugin-autotooltip_bigEnzyme: a macromolecule, usually a protein (but sometimes an RNA), that functions as a catalyst of some kind of biochemical reaction..

5’-G      AATTC-3’
   |          |
3’-CTTAA      G-5’

An enzymeplugin-autotooltip__default plugin-autotooltip_bigEnzyme: a macromolecule, usually a protein (but sometimes an RNA), that functions as a catalyst of some kind of biochemical reaction. called DNA ligaseplugin-autotooltip__default plugin-autotooltip_bigDNA ligase: an commercially available enzyme used to form phosphodiester bonds between the 3' end of one strand of dsDNA and the 5'end of another. Used for molecular cloning experiments. Its natural function in cells relates to lagging strand DNA synthesis and various DNA repair mechanisms. can then be used to re-form the phosphodiester bonds and close the circle to make an intact plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors..

Molecular cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of is extremely common in biological research and all the reagents commonly used for this process are easily available from a wide source of commercial suppliers. Here we have covered just the bare basics – there are all kinds of neat tricks that one can do when cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth.. Without going into further detail here, scientists now essentially have the capability to edit DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. at will using all of these tricks – if you can imagine it, any DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. can be created and cloned. If you are interested in some of the details you should take a molecular biologyplugin-autotooltip__default plugin-autotooltip_bigMolecular biology: the study of nucleic acids, specifically DNA and RNA. course or biotechnology practicum course, where these techniques will be discussed in much greater detail.

A collection of a large number of random chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. fragments ligated into plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. is known as a libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library.. Generation of a libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. yields a very large collection of plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors., each with a different chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. insert.

Say we wanted to cloneplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of the Lac operonplugin-autotooltip__default plugin-autotooltip_bigLac operon: an operon in E. coli that contains the cis-acting regulatory elements $lacP$ and $lacO$, and the protein coding genes $lacZ$, $lacY$, and $lacA$. The Lac operon is an example of a negatively-regulated operon., or genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) from the Lac operonplugin-autotooltip__default plugin-autotooltip_bigLac operon: an operon in E. coli that contains the cis-acting regulatory elements $lacP$ and $lacO$, and the protein coding genes $lacZ$, $lacY$, and $lacA$. The Lac operon is an example of a negatively-regulated operon. (more details on the Lac operonplugin-autotooltip__default plugin-autotooltip_bigLac operon: an operon in E. coli that contains the cis-acting regulatory elements $lacP$ and $lacO$, and the protein coding genes $lacZ$, $lacY$, and $lacA$. The Lac operon is an example of a negatively-regulated operon. are discussed in Chap. 10). First, a genomic libraryplugin-autotooltip__default plugin-autotooltip_bigGenomic library: a library generated by taking the genome of an organism, fragmenting the DNA, and cloning and archiving the collection of clones for research purposes. A good quality genomic library will have fragment sizes that are sufficient to encompass entire genes and will come close to representing the entire would be made from chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. from a Lac⁺ E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and using an R plasmidplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning. as our cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of vectorplugin-autotooltip__default plugin-autotooltip_bigVector: in molecular genetics, a vector is a genetic element (usually an episome of sort such as a plasmid but can be something like a virus) that is used to clone and/or transfer genes into and out of cells. (Fig. 11). To make this libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library., E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. (we can also say genomicplugin-autotooltip__default plugin-autotooltip_bigGenome: a dataset that contains all DNA information of an organism. Most of the time, this also includes annotation and curation of that information, e.g., the names, locations, and functions of genes within the genome. As an adjective (“genomic”), this usually is used in the context of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth.) is purified and cleaved with a restriction enzymeplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. to fragment the chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins.. An appropriate R plasmidplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning. is cleaved with the same restriction enzymeplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences., and the chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. fragments are ligated to the R plasmidplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning.. This libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. would then be used to transformplugin-autotooltip__default plugin-autotooltip_bigTransformation: in microbiology (including for yeasts), transformation is the alteration of phenotype due to uptake of external DNA into a cell. Not to be confused with “transformation” in the context of cell biology, where the definition is the alteration of a normal cell to a cancerous cell. a Lac^– E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and. Transformantsplugin-autotooltip__default plugin-autotooltip_bigTransformant: a bacterial or yeast cell that has been transformed with a foreign piece of DNA. (i.e., E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). cells carrying a plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. from the libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library.) would first be selected for by resistance to the antibioticplugin-autotooltip__default plugin-autotooltip_bigAntibiotics: naturally produced compounds, usually found in molds and fungi but are typically commercially available, that either kill bacteria or prevent their replication. In genetics research, they are often used to select for the presence of an episome that carries an antibiotic resistance ampicillinplugin-autotooltip__default plugin-autotooltip_bigAmpicillin: a common type of antibiotic used in genetics research., the phenotypeplugin-autotooltip__default plugin-autotooltip_bigPhenotype: an observable feature or property of an organism. (Amp^R) of which is conferred by a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) on the R plasmidplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning. we are using ($amp^r$). Any cell that is ampicillinplugin-autotooltip__default plugin-autotooltip_bigAmpicillin: a common type of antibiotic used in genetics research. resistant must carry a plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. from the libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library.. (As a side note, R plasmidsplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning. have a property that make it so that each cloneplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of can only carry one type of R plasmidplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning.; therefore, each cloneplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of will contain a different and unique chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. fragment.) The resistant coloniesplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of would then be selected for the ability to grow on lactose (Lac⁺). These clonesplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of should not only contain R plasmidsplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning., but they should also carry an insert that contains a functional Lac operonplugin-autotooltip__default plugin-autotooltip_bigLac operon: an operon in E. coli that contains the cis-acting regulatory elements $lacP$ and $lacO$, and the protein coding genes $lacZ$, $lacY$, and $lacA$. The Lac operon is an example of a negatively-regulated operon..

How many clonesplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of would we need to screenplugin-autotooltip__default plugin-autotooltip_bigScreen: a screen is a process through which a researcher looks through a population of individuals in an attempt to find rare individuals with certain phenotypes, usually with no obvious way to enrich for the rare individuals. contrast to a selection.? Depending on the type of restriction enzymeplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. used, each plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. carries about 4 x 10³ bp of chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. (the frequency of recognition sites is $(\frac{1}{4})^6$ for an enzymeplugin-autotooltip__default plugin-autotooltip_bigEnzyme: a macromolecule, usually a protein (but sometimes an RNA), that functions as a catalyst of some kind of biochemical reaction. like EcoRI that recognizes a 6 bp site, or one site every 4096 bp on average). The E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. is 5 x 10⁶ bp; thus, the entire E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). genomeplugin-autotooltip__default plugin-autotooltip_bigGenome: a dataset that contains all DNA information of an organism. Most of the time, this also includes annotation and curation of that information, e.g., the names, locations, and functions of genes within the genome. As an adjective (“genomic”), this usually is used in the context of will be covered if several thousand clonesplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of from the libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. are screened.

All sorts of genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) from E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). have been cloned by looking for DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. fragments that can restore function to a mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference.. It is also possible to find genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) from other bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably.. The following is a dramatic example of a cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of experiment to find an important proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. for a pathogenic bacteriumplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably..

Yersinia pestis is a bacteriumplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. that causes bubonic plague, a disease that killed 100 million people in the 6th century A.D. One reason that Yersinia is such a deadly pathogen is that it escapes the immune system by multiplying inside of cells. Scientists wanted to find the Yersinia genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that enable the bacterialplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. cells to invade human cells.

To do this an assay was needed – the scientists needed a way to experimentally observe bacterialplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. cells enter into mammalian cells. An assay for bacterialplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. invasion consists of a layer of cultured mammalian cells. The bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. are allowed to settle onto the cells for a while, then the bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. that have not entered the cells are killed with the antibioticplugin-autotooltip__default plugin-autotooltip_bigAntibiotics: naturally produced compounds, usually found in molds and fungi but are typically commercially available, that either kill bacteria or prevent their replication. In genetics research, they are often used to select for the presence of an episome that carries an antibiotic resistance gentamicin, which cannot cross the membrane of tissue culture cells. The bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. that have entered cells escape gentamicin and can be recovered from the inside of the cells after the mammalian cells are lysed with detergent.

E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). normally cannot invade mammalian cells. The geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) for bacterialplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. cellular invasion was found by creating a libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. of Yersinia DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. clonesplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of by cutting Yersinia chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. with restriction enzymesplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. and ligating the fragments into R plasmidsplugin-autotooltip__default plugin-autotooltip_bigR plasmid: a type of small bacterial high-copy number plasmid that is commonly used as a vector for molecular cloning.. The libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. was then transformedplugin-autotooltip__default plugin-autotooltip_bigTransformation: in microbiology (including for yeasts), transformation is the alteration of phenotype due to uptake of external DNA into a cell. Not to be confused with “transformation” in the context of cell biology, where the definition is the alteration of a normal cell to a cancerous cell. into E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). and then selectingplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a for E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). that had invaded tissue culture cells by killing the E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). outside of cells with gentamycin. A single geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) was found that encodes a surface proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. that became known as invasin.

There are lots of other molecular cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of tricks that scientists have developed in the last 50 years or so. The purpose of this chapter is not to discuss all the details of all possible cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of techniques (that would be enough material to be its own course!) but to show the overall idea of cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of and cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of by complementationplugin-autotooltip__default plugin-autotooltip_bigComplementation: a concept where an additional allele of a gene (usually a wildtype allele) can provide normal function to an organism with a recessive loss of function mutation in that gene. The concept of complementation underlies the complementation test.. Current molecular cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of technology allows scientists to custom build essentially any DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. they desire.

Exercise 1: In Chap. 05 we discussed the relationship between recombinationplugin-autotooltip__default plugin-autotooltip_bigRecombination: Recombination can have slightly different meanings depending on context:

* In the context of genetic crosses (usually a dihybrid cross or a test cross), recombination refers to the phenomena where the phenotype of the F2 offspring is different than either parent (P generation). $lox$ frequency (m.u. or cMplugin-autotooltip__default plugin-autotooltip_bigMap unit (m.u.): a unit of genetic distance defined as 1% recombination. Also called a centiMorgan or cM.) and physical distance (measured in bp of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth.) (see also Chap. 12 Fig. 1). What is the relationship between minutes in Hfrplugin-autotooltip__default plugin-autotooltip_bigF: “F” can have several different related definitions:

* An F plasmid is a type of naturally occurring low copy number plasmid in E. coli that can move from one host cell to another via conjugation, a type of sex in bacteria (“F” stands for fertility). F plasmids mappingplugin-autotooltip__default plugin-autotooltip_bigGenetic mapping: a term describing a variety of different experimental approaches used to determine the physical locations of genes on chromosomes. and physical distance? In other words, how many bp of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. is equivalent to 1 minute? See Chap. 08 for some key information you might need.

Conceptual question: why would a strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and with a mutated modifying enzymeplugin-autotooltip__default plugin-autotooltip_bigEnzyme: a macromolecule, usually a protein (but sometimes an RNA), that functions as a catalyst of some kind of biochemical reaction. but a wildtypeplugin-autotooltip__default plugin-autotooltip_bigWildtype: a reference strain of an organism that scientists operationally define as “normal” to which mutants are compared. Not to be confused with wild organisms. restriction enzymeplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. ($R^+ M^-$) be inviable (incompatible with life)? In other words, why is $R^+ M^-$ a lethal combination of allelesplugin-autotooltip__default plugin-autotooltip_bigAllele: a version of a gene. Alleles of a gene are different if they have differences in their DNA sequence.?