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Microbiology: Chapter 11 Genetic Engineering and Biotechnology
Terms in this set (80)
The use of in vitro techniques in the isolation, alteration, and expression of DNA or RNA and in the development of genetically modified organisms. Altered genes may be reinserted into the original source organism or into some other host organism. Requires that DNA be isolated
A major class of enzymes that recognizes a specific DNA sequence and then cuts the phosphodiester backbone of the DNA; also known as restriction endonucleases. Widespread among Bacteria and Archaea, but very rare in eukaryotes. Several thousand with different specificites are known.
Type I & III
Restriction enzymes that bind to the DNA at their recognition sequences but cut the DNA at some distance away.
Restriction enzymes that cleave the DNA within their recognition sequences, making this class of enzymes more useful for the specific manipulation of DNA. Most sequences recognized by these enzymes are short inverted repeats of 4 to 8 bp.
inverted repeat sequences of 4 to 8 bp.
Type of staggered cut made by an endonuclease which leaves short, single-stranded overhangs at the ends of the two fragments.
Type of cut made by an endonuclease in which both strands are cut opposite each other.
Enzymes that chemically alter bases within a restriction enzyme recognition site and thus prevent the site from being cut. Enable a cell to protect its own DNA from inadvertent destruction by its own restriction enzymes. Typically, this consists of methylating specific bases within the recognition sequence which prevents the endonuclease from binding, thus these enzymes are called methylases.
a technique for separation of nucleic acid molecules based on size by passing an electric current through a gel made of agarose or polyacrylamide. The nucleic acids move through the gel toward the positive electrode due to their negatively charged phosphate groups. Small molecules migrate more rapidly than large. The rate of migration is determined by the size, shape and charge on the molecule. Also used to verify amplification.
The formation of a double helix by the base pairing of single strands of DNA or RNA from two different (but related) sources. It is used in detecting, characterizing, and identifying segments of DNA and RNA. It can be very useful for finding related sequences in different genomes or other genetic elements and to determine if a gene is expressed into an RNA transcript.
nucleic acid probes or probes
Segments of single-stranded nucleic acids whose identity is already known and that are used in hybridization. A strand of nucleic acid that can be labeled or made radioactive and used to hybridize to a complementary molecule from a mixture of nucleic acids.
A hybridization procedure where DNA is the target and RNA or DNA is the probe. The DNA fragments are first denatured to yield single strands and then transferred to a synthetic membrane. The membrane is then exposed to a labeled probe. If complementary to any fragments, hybrids form.
A hybridization procedure where RNA is the target and DNA or RNA is the probe. Often used to identify messenger RNA (mRNA) derived from specific genes. Intensity of the blot gives a rough estimate of mRNA abundance and thus can be used to monitor transcription.
Fluorescnce in situ hybridization (FISH)
A hybridization technique in which a range of different fluorescent signals can be covalently bonded to oligonucleotide (short single-stranded DNA or RNA molecules) probes to target specific DNA sequences. It can be used to identify strains of bacteria by hybridizing to characteristic sequences in the genes for their 16S ribosomal RNA, which vary slightly from strain to strain.
hybridization using replica plating
A hybridization procedure in which total genomic DNA is cloned and hybridization on the resulting colonies using a nucleic acid probe can detect recombinant DNA in colonies. In this procedure a duplicate of the master plate is made on a membrane filter. It can be used to detect the presence of specific genes in genomes as well as movement of genetic elements.
The process by which multiple copies of DNA are formed.
polymerase chain reaction (PCR)
Artificial amplification of a DNA sequence by repeated cycles of strand separation and replication. It yields large amounts of specific genes or other DNA segments that may be used for a range of applications in molecular biology. It uses the enzyme DNA polymerase, which naturally copies DNA molecules and artificially synthesized primers made of DNA.
A relatively short, single-stranded fragment of RNA or DNA molecule with a defined sequence (chemical structure) that is complementary to a chosen sequence.
An automated PCR machine used to create large amounts of amplified DNA.
Taq and Pfu polymerases
Two thermostable DNA enzymes used in the PCR due to their being unaffected by the high temperatures used in the denaturation of the double stranded copies of DNA in vitro.
Reverse transcription RT-PCR
A variation in the standard PCR procedure in which DNA is made from an mRNA template using the retroviral enzyme reverse transcriptase to convert RNA into complementary DNA (cDNA). A primer complementary to the 3' end (poly(A) tail in eukaryotic cells) is used. A hybrid molecule containing both RNA & DNA is produced as an intermediate. The RNA is then degraded by RNase H leaving single-stranded cDNA as a template for standard PCR. Can be used to detect if a gene is expressed or to produce an intron-free eukaryotic gene.
quantitative PCR (qPCR)
A variation in the standard PCR procedure which quantifies the amount of initial target DNA or RNA in a sample by using fluorescent probes to monitor the amplification process.
The isolation and incorporation of a fragment of DNA into a vector where it can be replicated.
(as in cloning) a self-replicating DNA molecule that is used to carry cloned genes or other DNA segments for genetic engineering. A small, simple, and manipulable genetic element (e.g. plasmid, virus) that can independently replicate that is used to carry and replicate a desired gene or segment of DNA that has moved from its original location.
A DNA molecule containing DNA originating from two or more sources. When it replicates, the cloned DNA that it contains is also replicated.
isolation and fragmentation of the source DNA
The first step in gene cloning. The source DNA can be:
- total genomic DNA (must be cut with restriction enzymes)
- DNA synthesized from RNA
- a gene or amplified genes (by PCR)
- synthetic DNA made in vitro
inserting the DNA fragment into a cloning vector
The second step of gene cloning in which a foreign DNA fragment is joined to a cloning vector, typically a plasmid or virus. This is done at a restriction site on the vector using restriction enzymes and DNA ligase, an enzyme that covalently links both strands of the vector and the foreign DNA.
introduction of the cloned DNA into a host organism
The third step of gene cloning involves the introduction of the recombinant DNA made in vitro into a host organism where it can replicate. This is often done through transformation. Often yields a mixture of recombinant constructs - some cells contain the desired cloned gene, whereas other cells may contain other cloned genes from the same source DNA.
genomic library (DNA library)
A collection of cloned DNA segments that is big enough to contain at least one copy of every gene from a particular organism.
Making a genomic library by random cloning of DNA fragments.
This marker can be selected to isolate host cells containing a plasmid vector so that only these cells form colonies.
To isolate host cells containing a viral vector, this particular type of colony can be found.
selections and screenings
When cloning a single DNA fragment generated by PCR or other purified means, these testing methods are usually sufficient to identify the host cells carrying the cloned DNA.
DNA sequencing, restriction digests on plasmids, hybridization
These methods can be used to isolate host cells containing the gene of interest when the cells contain many other cloned genes or a genomic library.
These can be used to identify colonies that contain a cloned gene that encodes a protein that has been expressed in the cloning host, by binding specifically with the protein. This encoded protein is the antigen. The host itself must not produce the protein. Replica plating and a radioactive reagent that binds to these are used. These must be made in an experimental animal against the antigen (protein) of interest.
Construction in vitro of a gene with a specific mutation at a precisely determined site using synthetic DNA plus DNA cloning techniques. By altering gene sequences to produce amino acid sequence changes, this is used to manipulate protein characteristics such as enzyme activity or protein-binding affinity.
This can be used as a primer or probe for PCR, in-site mutagenesis, hybridization, or to provide altered version of parts of genes or regulatory regions. Oglionucleotides can be over 100 bp in length (typical is 12-40).
DNA cassettes or cartridges
An artificially designed segment of DNA that usually carries a gene for resistance to an antibiotic or some other convenient marker and is flanked by convenient restriction sites. When used to REPLACE sections of genes, they are typically the same size as the wild-type fragments that they replace. Can be any size when used for making INSERTION mutations (gene disruptions).
Creating mutations by the insertion of a DNA cassette. Used to make more than a few base-pair changes or replace sections of a gene of interest. The cassette can REPLACE sections of the DNA of interest by:
- using restriction sites, if present at the required location
- site-directed mutagenesis
gene disruption or gene knockout
A type of cassette mutagenesis in which cassettes are inserted into the middle of a gene, thus disrupting the coding sequence. The inactivation of a gene by insertion of a DNA fragment that interrupts the coding sequence. Inserted cassettes that carry a gene for resistance to an antibiotic cause the host cell to gain antibiotic resistance. Similar to mutations made by transposons, but experimenter chooses gene that will be mutated. Used to determine whether a gene is essential in prokaryotes (haploid).
A gene used in genetic analysis because the product it encodes, a protein, is easy to detect.. They can be used for the following purposes:
- report the presence of a particular genetic element (like plasmid)
- report the abscence of a particular genetic element
- report the presence of DNA inserted in a vector
- can be fused to other genes or to the promoter of other genes so
gene expression can be studied
green fluorescent protein (GFP)
A protein that glows green and is widely used in genetic analysis as a reporter gene.
A structure created by joining together segments of two separate genes. In particular, when the regulatory region of one gene (gene of interest) is joined to the coding region of a reporter gene. The reporter is then made under the conditions that would trigger expression of the target gene. It is used in studying gene regulation, especially if measuring the levels of the natural gene product is difficult, expensive, or time consuming.
A gene fusion in which a coding sequence that retains its own translational signals is fused to the transcriptional signals of another gene.
A gene fusion in which two coding sequences are fused so that they share the same transcriptional and translational start and stop signals. Following translation, protein fusions yield a single hybrid polypeptide.
Assayed by fusing the transcriptional start signals of a gene of interest to a reporter gene.
Assayed by fusing translational start signals of a gene of interest to a reporter gene under the control of a known promoter.
The use of an electric pulse to enable cells to take up DNA.
chemically mediated transformation
the transfer of bacterial genes involving free DNA and chemicals. The free DNA binds to the cell surface by DNA binding proteins. Then either both strands or one strand is taken-up. It is then bound by a protein to protect it from nuclease attack until it reaches the chromosome where it is integrated by recombination.
Plasmid that was derived from a ColE1 (E. coli) toxin-encoding plasmid by removing the toxin genes and inserting ampicillin resistance and a blue-white color-screening system. It contains a lacZ gene which encodes the lactose-degrading enzyme beta-galactosidase. Into this lacZ gene, a short segment of artificial DNA containing cut sites for many restriction enzymes was inserted. It is a widely used cloning vector.
multiple cloning site (MCS)
The short segment of DNA containing cut sites for many restriction enzymes in the lacZ gene of the pUC19 plasmid. Treatment with particular restriction enzymes opens the vector at a unique (and only one) location. It does not inactivate lacZ.
the disruption of the lacZ gene in plasmid pUC19 when foreign DNA is inserted into the gene. It is used to detect the presence of foreign DNA within the vector or recombinant vector.
A colorless reagent used to test for beta-galactosidase activity, which cleaves to it, generating a blue product.
Bacteria: Escherichia coli & bacillus subtilis
Eukaryote: Saccharomyces cerevisiae (yeast)
The most useful hosts for cloning. These microorganisms are easily grown and we have considerable background information on them and a wealth of tools for their genetic manipulation. Complete genome sequences are available for them.
This bacterial host has the following advantages as a host for cloning:
- Well-developed genetics
- many strains available
- potentially pathogenic
- has outer membrane that hinders protein secretion
- lacks systems to correctly modify eukaryotic proteins (any bacterial
This bacteria has the following advantages as a host for cloning:
- easily transformed
- naturally secretes proteins (gram-positive)
- endospore formation simplifies culture
- genetically unstable
- plasmid instability
- foreign DNA not well maintained, cloned DNA often lost
S. crevisiae (yeast)
This eukaryote has the following advantages as a host for cloning:
- well developed genetics
- possess complex RNA and post-translational processing systems
required for the production of eukaryotic proteins
- east to grow
- plasmids unstable
- won't replicate most bacterial plasmids
Gene cloning has been used on these cells to do research on human genetics, cancer, infectious disease, and physiology. Disadvantages are that they are expensive to use and difficult to produce under large-scale conditions. Since eukaryotic cells don't exhibit transformation, translation, and conjugation. The transfer of DNA into eukaryotic cells includes transfection, microinjection, and electroporation.
A cloning vector that can replicate in two or more dissimilar hosts. Genes carried by this vector can be moved between unrelated organisms.
yeast origin of replication, centromere recognition sequence (CEN sequence)
In order for a bacterial (E. coli) plasmid vector to be shuttled to a eukaryote (yeast), these two modifications must be made to the plasmid: addition of ______________ & a segment of DNA from the yeast needed for proper cell division called the _____________.
A cloning vector that contains the necessary regulatory sequences to allow transcription and translation of cloned genes. Usually the control is transcriptional because for high levels of expression it is essential to produce high levels of mRNA. Thus, a strong promotor that binds RNA polymerase is required.
lac (the lac operon promoter)
trp (the trp operon promoter)
tac & trc (synthetic hybrids of the trp & lac promoters)
Strong promoters in E. coli that can be specifically regulated.
T7 promoter & gene that enodes T7 RNA polymerase
T7 expression vectors, used for generating large amounts of a particular protein, require these two components of the T7 bacteriophage to control gene expression. One is integrated into the plasmid vector (pET plasmid for BL21 series of E. coli) containing the cloned gene, and the other is integrated into the chromosome of the host (typically E. coli).
Cosmids, artificial chromosomes, bacteriophages, and derivatives of viruses (SV40 virus (polyomavirus), adenovirus, vaccinia (pox virus), baculovirus, retroviruses)
Plasmid vectors used for molecular cloning are limited in the amount of DNA that can be inserted, with 10kbp being the max. When cloning large genomic regions such as operons and eukaryotic genes these vector types are needed. DNA is directly integrated into the host.
This bacteriophage serves as a useful cloning vector since a third of its genome can be replaced with foreign DNA. In order to use it as a cloning vector its restriction enzyme sites and a multiple cloning site (MCS) containing the gene for beta-galactosidase (lacZ) is added to select for vectors that contain recombinant DNA.
Plating with an E. coli strain using an Xgal-containing agar plate and screening for interruption of the beta-galactosidase gene. Clear plaques have cloned DNA, blue plaques do not.
When using bacteriophage lambda as a cloning vector, individual clones can be isolated in this way.
Plasmid vectors containing the cos site from the lambda genome, which yields cohesive ends when cut. The cos site is required for packaging DNA into lambda virions. Constructed from ligating the lambda cos region to plasmid DNA; foreign DNA is then ligated to the vector. This plasmid with cloned DNA can then be packaged into lambda virions in vitro. These virions can be used to transduce E. coli. The foreign DNA can consist of large fragments (50 kpb).
A single copy vector that can carry extremely long inserts of DNA and is widely used for cloning segments of large genomes. Used for making libraries of DNA from eukaryotic microorganisms or from higher eukaryotes such as humans. Allows the size of the library to be manageable.
bacterial artificial chromosomes (BAC's)
A cloning vector that consists of a circular artificial chromosome with bacterial origin of replication (required). Typically constructed from the F plasmid, and the host is generally a mutant strain of E. coli. Foreign DNA of more than 300 kpb can be inserted into this type of vector.
yeast artificial chromosome (YAC's)
A cloning vector that is an artificial chromosome with the following requirements to function like normal eukaryotic chromosomes:
1) yeast origin of DNA replication
2) telomeres at the ends of the of the chromosomes for replication
3) a centromere sequence (CEN) for segregation during mitosis
4) a cloning site - need restriction site
5) a gene for selection (selectable marker) following transformation
into the host
Host is typically the yeast S. cerevisiae. Foreign DNA of up to 800 kpb can be inserted.
The use of organisms, typically genetically altered, in industrial, medical, or agricultural applications.
1) the eukaryotic genes must be placed under control of a
2) any introns must be removed from the gene (DNA)
3) codon bias may require edits to gene sequences
4) many mammalian proteins require modification after
translation to yield the active form
Obstacles to cloning a mammalian gene in bacteria.
reverse transcription PCR
The usual way to obtain an intron-free eukaryotic gene is to clone its mRNA. Transcription of the eukaryotic gene and processing results in mature mRNA, with no introns. This process is used after mRNA, which carries an uninterrupted coding sequence for a particular gene, has been isolated to convert it to complementary DNA (cDNA). This double-stranded cDNA contains the coding sequence but lacks introns, allowing it to be inserted into a plasmid or other vector for cloning. cDNA contains only the DNA sequences encoding the protein, it lacks a promoter and other upstream regulatory sequences necessary for expression. Thus, special expression vectors with bacterial promoters and ribosome-binding sites are needed when genes are cloned this way.
If the amino acid sequence of a desired protein is known, it can be used to design and synthesize an _____________ that encodes it. Due to the degenerate code, many of these are possible for a polypeptide sequence. If codon usage of the target organism is known, a preferred sequence can be selected. Only a region of the gene is necessary since this will be nearly complementary to the target mRNA of the gene of interest and this is will be used as a probe.
A plant or an animal with foreign DNA inserted into its genome.
genetically modified organisms (GMOs)
An organism whose genome has been altered using genetic engineering; the abbreviation GM is also used in terms such as GM crops and GM foods.
A vaccine made by inserting genes from a pathogenic virus into a relatively harmless carrier virus. These induce immunity to the pathogenic virus.
A vaccine that immunizes against more than one disease.
A carrier virus that is widely used to prepare recombinant vector vaccines (contains recombinant nucleic acid in which genes that encode for virulence factors are deleted from a pathogenic bacterium or virus, but those whose products elicit an immune response are left) for human use. The virus itself is not pathogenic for humans and has been used as a vaccine against the related smallpox virus. Many vaccines have been developed.
A vaccine that contains only a specific protein or two from a pathogenic organism. For a pathogenic virus, this is often the coat protein because coat proteins are highly immunogenic. The coat proteins are purified and used in high dosage to elicit a rapid and high level of immunity without containing the intact pathogen. Sometimes poorly immuonogenic when produced in bacteria. More effective if a eukaryotic cloning host (yeast) is used.
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