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

Figure 9.1.1

Overview

Figure 9.1.2

Table 9.1.1

Table 9.1.2

• (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

Tools of Biotechnology

Figure 9.2

Restriction Enzymes

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

• 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)

Tools of Biotechnology

Vectors

Figure 9.3

• 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

Tools of Biotechnology

PCR

Figure 9.4.1

PCR

Figure 9.4.2

Figure 9.5b

Techniques of Genetic Engineering

• Inserting foreign DNA into cells– Transformation– Electroporation– Protoplast fusion– Gene gun– Microinjection

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

Figure 9.6 & 7

Techniques of Genetic Engineering

• Microinjection: introduce DNA directly into an animal cell using a glass miropipette

Chapter 9

Biotechnology and Recombinant DNA

Part 2

• 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

Tools of Biotechnology

Figure 9.2

Restriction Enzymes

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

• 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)

Tools of Biotechnology

Vectors

Figure 9.3

• 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

Tools of Biotechnology

PCR

Figure 9.4.1

PCR

Figure 9.4.2

Figure 9.5b

Techniques of Genetic Engineering

• Inserting foreign DNA into cells– Transformation– Electroporation– Protoplast fusion– Gene gun– Microinjection

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

Figure 9.6 & 7

Techniques of Genetic Engineering

• Microinjection: introduce DNA directly into an animal cell using a glass miropipette

• 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

Obtaining DNA

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

Figure 9.11.1

Blue-white screening

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

Figure 9.12.1

Colony hybridization

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

• 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

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

• 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

Therapeutic applications

Random Shotgun Sequencing

Figure 9.14

• Understanding of DNA

• Sequencing organisms' genomes

• DNA fingerprinting for identification

Scientific Applications

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

• 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?

Safety Issues and Ethics

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– 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)