Biotechnology and Recombinant DNA Chapter 9 Biotechnology and Recombinant DNA

Introduction to Biotechnology Biotechnology: use of microorganisms, cells, or cell components to make a product Foods, antibiotics, vaccines, vitamins, enzymes Recombinant DNA Technology: Insertion or modification of genes to produce desired products (genes or proteins); manufacturing and manipulating genetic material in vitro also called genetic engineering

Overview Figure 9.1.1

Figure 9.1.2

Table 9.1.1

Table 9.1.2

Tools of Biotechnology (Artificial) Selection: Culture a naturally-occurring microbe that produces desired product Isolate bacteria and fungi from nature & use pure culture technique Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait Site-directed mutagenesis: Change a specific DNA code to change a protein Select and culture microbe with the desired mutation

Tools of Biotechnology Restriction Enzymes DNA cutting enzymes that exist in many bacteria Cut specific sequences of DNA (recognize 4-, 6-, or 8-base sequences), staggered cuts Destroy bacteriophage DNA in bacterial cells Cannot digest (host) DNA with methylated cytosines

Restriction Enzymes Figure 9.2

Tools of Biotechnology Vectors Carry new DNA to desired cell Plasmids and viruses can be used as vectors Four properties of vectors Can self-replicate Be a size that allows them to be manipulated outside the cell during recombinant DNA procedures Preservation (circular form of DNA and integrated into host chromosome) Have a marker within the vector for easy selection

Tools of Biotechnology Shuttle vectors: a plasmid that can exist in several different species Very useful in the process of genetically modifying multicellular organisms Viral DNA can usually accept much larger pieces of foreign DNA than plasmid Retroviruses, adenoviruses, & herpesviruses Choice of suitable vector depends on many factors (e.g host & size of the DNA to be cloned)

Vectors Figure 9.3

Tools of Biotechnology Polymerase Chain Reaction (PCR) To make multiple copies of a piece of DNA enzymatically (limited by the choice of primers used) Cannot be used to amplify an entire genome Used to Clone DNA for recombination Amplify DNA to detectable levels Sequence DNA Diagnose genetic disease Detect pathogens

PCR Figure 9.4.1

PCR Figure 9.4.2

Techniques of Genetic Engineering Inserting foreign DNA into cells Transformation Electroporation Protoplast fusion Gene gun Microinjection Figure 9.5b

Techniques of Genetic Engineering Choice of method is usually determined by the type of vector and host being used Foreign DNA will survive only if it is either present on a self-replicating vector or incorporated into one of the cell’s chromosomes by recombination

Techniques of Genetic Engineering Transformation: used to insert plasmid vector into a cell many cell types do not naturally transform need to make them competent (able to take up external DNA) Electroporation: uses an electrical current to form microscopic pores in the membranes of cells (DNA enter cells through the pores)

Techniques of Genetic Engineering Generally applicable to all cells; ones with cell wall must be converted to protoplasts first Protoplast fusion: a method of joining two cells by first removing their cell walls Protoplasts in solution will fuse at a low but significant rate (can add polyethylene glycol to increase the frequency of fusion) Valuable in the genetic manipulation of plant and algal cells

Techniques of Genetic Engineering Gene gun: Microscopic particles of tungsten or gold are coated with DNA and propelled by a burst of helium through the plant cell walls Some of the cells express the introduced DNA as if it were their own if incorporated into host chromosome

Techniques of Genetic Engineering Microinjection: introduce DNA directly into an animal cell using a glass miropipette Figure 9.6 & 7

Biotechnology and Recombinant DNA Part 2 Chapter 9 Biotechnology and Recombinant DNA Part 2

Tools of Biotechnology Restriction Enzymes DNA cutting enzymes that exist in many bacteria Cut specific sequences of DNA (recognize 4-, 6-, or 8-base sequences), staggered cuts Destroy bacteriophage DNA in bacterial cells Cannot digest (host) DNA with methylated cytosines

Restriction Enzymes Figure 9.2

Tools of Biotechnology Vectors Carry new DNA to desired cell Plasmids and viruses can be used as vectors Four properties of vectors Can self-replicate Be a size that allows them to be manipulated outside the cell during recombinant DNA procedures Preservation (circular form of DNA and integrated into host chromosome) Have a marker within the vector for easy selection

Tools of Biotechnology Shuttle vectors: a plasmid that can exist in several different species Very useful in the process of geneticaly modifying multicellular organisms Viral DNA can usually accept much larger pieces of foreign DNA than plasmid Retroviruses, adenoviruses, & herpesviruses Choice of suitable vector depends on many factors (e.g host & size of the DNA to be cloned)

Vectors Figure 9.3

Tools of Biotechnology Polymerase Chain Reaction (PCR) To make multiple copies of a piece of DNA enzymatically (limited by the choice of primers used) Cannot be used to amplify an entire genome Used to Clone DNA for recombination Amplify DNA to detectable levels Sequence DNA Diagnose genetic disease Detect pathogens

PCR Figure 9.4.1

PCR Figure 9.4.2

Techniques of Genetic Engineering Inserting foreign DNA into cells Transformation Electroporation Protoplast fusion Gene gun Microinjection Figure 9.5b

Techniques of Genetic Engineering Choice of method is usually determined by the type of vector and host being used Foreign DNA will survive only if it is either present on a self-replicating vector or incorporated into one of the cell’s chromosomes by recombination

Techniques of Genetic Engineering Transformation: used to insert plasmid vector into a cell many cell types do not naturally transform need to make them competent (able to take up external DNA) Electroporation: uses an electrical current to form microscopic pores in the membranes of cells (DNA enter cells through the pores)

Techniques of Genetic Engineering Generally applicable to all cells; ones with cell wall must be converted to protoplasts first Protoplast fusion: a method of joining two cells by first removing their cell walls Protoplasts in solution will fuse at a low but significant rate (can add polyethylene glycol to increase the frequency of fusion) Valuable in the genetic manipulation of plant and algal cells

Fig. 9.5

Techniques of Genetic Engineering Gene gun: Microscopic particles of tungsten or gold are coated with DNA and propelled by a burst of helium through the plant cell walls Some of the cells express the introduced DNA as if it were their own if incorporated into host chromosome

Techniques of Genetic Engineering Microinjection: introduce DNA directly into an animal cell using a glass miropipette Figure 9.6 & 7

Obtaining DNA Gene library: a collection of cloned DNA fragments created by inserting restriction enzyme fragments in a bacterium, yeast, or phage Make a collection of clones large enough to ensure that at least one clone exists for every gene in the organism Pieces of an entire genome stored in plasmids or phage

Fig. 9.8

Obtaining DNA Cloning genes from eukaryotic organisms poses a special problems due to introns Need to use a version of the genes that lacks intron = mRNA cDNA is made from mRNA by reverse transcriptase (mRNA cDNA) cDNA is the most common method of obtaining eukaryotic genes

Fig. 9.9

Obtaining DNA Synthetic DNA is made by a DNA synthesis machine Chain of over 120 nucleotides can be synthesized Need to know the sequence of the gene Rare to clone a gene by synthesizing it directly Plays a much more useful role selection procedures (add desired restriction sites)

Selecting a clone Use antibiotic resistance genes (marker) on plasmid vectors to screen for cells carrying the desired gene (engineered vector) e.g. Blue-white screening (2 marker genes on the plasmid vector = ampR and -galactosidase)

Genetic Engineering Blue-white screening Figure 9.11.1

Genetic Engineering Figure 9.11.2

Selecting a clone Need a second procedure to test if screened bacteria does contain desirable genes Test clones for desired gene product or ID genes itself in the host bacterium Colony hybridization: use DNA probe that is complementary to the desired genes DNA probe: short segment of single-stranded DNA that are complementary to the desired gene

Colony hybridization Figure 9.12.1

Colony hybridization Figure 9.12.2

Making a gene product Earliest work in genetic engineering used E. coli to synthesize the gene products E. coli was used because it is easily grown and its genomics are known Disadvantages of using E. coli: Produce endotoxins (Lipid A, part of LPS layer on the cell wall) Does not secrete protein products need to lyse cells to obtain products Industry prefers Bacillus subtilis because it secretes their products

Making a gene product Use baker’s yeast (Saccharomyces cerevisiae) Yeast may carry plasmid and has best understood eukaryotic genome May be more successful in expressing foreign eukaryotic genes than bacteria; likely to secrete products Use mammalian cells in culture Hosts for growing viruses (vectors) Often the best suited to making protein products for medical use (e.g. hormones, cytokines, interferon)

Making a gene product Use plant cells in culture Ti plasmid (from bacterium Agrobacterium tumefaciens), protoplast fusion and gene gun Use to produce genetically engineered plants May be sources for plant alkaloids (painkiller), isoprenoids (basis for synthetic rubber), and melanin (for sunscreens)

Applications of Genetic Engineering Produce useful substances more efficiently and cheaper Obtain information from the cloned DNA that is useful for either basic research or medical applications Use cloned genes to alter the characteristics of cells or organisms

Therapeutic applications Subunit vaccines Nonpathogenic viruses carrying genes for pathogen's antigens as vaccines Gene therapy to replace defective or missing genes Human Genome Project Nucleotides have been sequenced Human Proteome Project may provide diagnostics and treatments

Random Shotgun Sequencing Figure 9.14

Scientific Applications Understanding of DNA Sequencing organisms' genomes DNA fingerprinting for identification Figure 9.16

Southern Blotting Figure 9.15.1

Southern Blotting Figure 9.15.2

Southern Blotting Figure 9.15.3

Agricultural Applications Table 9.2

Genetic Engineering Using Agrobacterium Figure 9.18

Safety Issues and Ethics Avoid accidental release Genetically modified crops must be safe for consumption and for the environment Who will have access to an individual's genetic information?

Chapter Review: Biotechnology Use of microorganisms, cells, or cell components to make a product Organisms that has desirable traits can be obtained by 1) selection (aritificial) or 2) mutation (if you know a specific DNA code you want, you can use site-directed mutagenesis to create a mutant)

Biotechnology Restriction enzymes Vectors (plasmids and viruses) DNA cutting enzymes that cut specific sequence of double-stranded DNA (staggered cuts) Used to cut out the genes of interest (DNA fragment) and insert the gene into a vector Vectors (plasmids and viruses) vehicle to carry new DNA to a desired host cell

Biotechnology Vectors Properties: 1) self-replicating, 2) Be a size that allows them to be manipulated outside the cell (recombinant DNA procedure step), 3) preservation, and 4) have a marker for easy detection shuttle vectors used to deliver DNA fragments to several different species (esp. useful for multicellular organisms.)

Biotechnology Vectors Virus can accept much larger foreign DNA fragment Choice of suitable vector depends on the host and the size of the DNA fragment to be cloned

Biotechnology Polymerase Chain Reaction (PCR) To make multiple copies of a DNA fragment; not for amplifying entire genome Used for 1) cloning DNA for recombination, 2) amplify DNA to detectable levels, 3) DNA sequencing, 4) Diagnose genetic disease, 5) Detect pathogens

Genetic Engineering/ Recombinant DNA Technology Insertion or modification of genes to produce desired products (genes or proteins); manufacturing and manipulating genetic material in vitro Isolate or obtain DNA with desired gene Gene library: a collection of cloned DNA fragments created by inserting restriction enzyme fragments in a bacterium, yeast, or phage In eukaryotes, use cDNA (made from mRNA & reverse transcriptase) instead of DNA Synthetic DNA

Genetic Engineering/ Recombinant DNA Technology Insert foreign DNA into a host cell Trasformation: Used to insert plasmid vector into a cell (many cells are not naturally competent to be transformed) Electroporation: uses an electrical current to form microscopic pores in the membranes of cells & DNA enters cells through the pores (generally applicable to all cells)

Genetic Engineering/ Recombinant DNA Technology Protoplast fusion: a method of joining two cells by first removing their cell walls (valuable for plant and algal cells) Gene gun: microscopic particles of tungsten or gold coated with DNA and shot through plant cell walls by a burst of helium Microinjection: Introduce DNA directly into an animal cell using a glass micropipette

Genetic Engineering/ Recombinant DNA Technology Host cells to make a gene product Earliest work done using E. coli (plasmid vector) Baker’s yeast (plasmid vector), eukaryotic cells Mammalian cells in culture (virus vectors, electroporation) --> best suited to make protein products for medical use Plant cells in culture (Ti plasmid, protoplast fusion, & gene gun) --> genetically engineered plants

Genetic Engineering/ Recombinant DNA Technology Selecting or finding a clone which contains the recombinant vector Blue-white screening Colony hybridization

Application of Genetic Engineering Therapeutic application Subunit vaccines --> no chance of becoming infected from the vaccine Gene therapy --> replace defective or missing genes with correct genes Human genome project may provide diagnostics and treatments

Application of Genetic Engineering Scientific application Understand DNA; sequence genome; DNA fingerprinting for identifying bacterial/viral pathogens; forensic medicine (paternity, & evidence for a crime) Agricultural application Produce genetically engineered plants (more yield per plant, resistant to herbicide, insects, etc.)

Chapter Review Know these terms: biotechnology, genetic engineering/recombinant DNA technology, transformation, electroporation, protoplast fusion, gene gun, and microinjection Know Fig. 9.1 Know how these tools of biotechnology are used: selection vs. mutation, restriction enzymes, vectors, and PCR (not procedures)

Chapter Review Know the use of gene library, and ways to select a host which has picked up the recombinant (engineered) vector You do not have to know the procedures for making gene library Know therapeutic application, human genome project, and agricultural application (from the review slides)