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Page 1: Ch13 lecture biotechnology

Biotechnology

13

Page 2: Ch13 lecture biotechnology

Chapter 13 Biotechnology

Key Concepts

• 13.1 Recombinant DNA Can Be Made in the Laboratory

• 13.2 DNA Can Genetically Transform Cells and Organisms

• 13.3 Genes and Gene Expression Can Be Manipulated

• 13.4 Biotechnology Has Wide Applications

Page 3: Ch13 lecture biotechnology

Chapter 13 Opening Question

How is biotechnology used to alleviate environmental problems?

Page 4: Ch13 lecture biotechnology

Concept 13.1 Recombinant DNA Can Be Made in the Laboratory

It is possible to modify organisms with genes from other, distantly related organisms.

Recombinant DNA is a DNA molecule made in the laboratory that is derived from at least two genetic sources.

Page 5: Ch13 lecture biotechnology

Concept 13.1 Recombinant DNA Can Be Made in the Laboratory

Three key tools:

• Restriction enzymes for cutting DNA into fragments

• Gel electrophoresis for analysis and purification of DNA fragments

• DNA ligase for joining DNA fragments together in new combinations

Page 6: Ch13 lecture biotechnology

Concept 13.1 Recombinant DNA Can Be Made in the Laboratory

Restriction enzymes recognize a specific DNA sequence called a recognition sequence or restriction site.

5′…….GAATTC……3′

3′…….CTTAAG……5′

Each sequence forms a palindrome: the opposite strands have the same sequence when read from the 5′ end.

Page 7: Ch13 lecture biotechnology

Figure 13.1 Bacteria Fight Invading Viruses by Making Restriction Enzymes

Page 8: Ch13 lecture biotechnology

Concept 13.1 Recombinant DNA Can Be Made in the Laboratory

Some restriction enzymes cut DNA leaving a short sequence of single-stranded DNA at each end.

Staggered cuts result in overhangs, or “sticky ends;” straight cuts result in “blunt ends.”

Sticky ends can bind complementary sequences on other DNA molecules.

Methylases add methyl groups to restriction sites and protect the bacterial cell from its own restriction enzymes.

Page 9: Ch13 lecture biotechnology

Concept 13.1 Recombinant DNA Can Be Made in the Laboratory

Many restriction enzymes with unique recognition sequences have been purified.

In the lab they can be used to cut DNA samples from the same source.

A restriction digest combines different enzymes to cut DNA at specific places.

Gel electrophoresis analysis can create a map of the intact DNA molecule from the formed fragments.

Page 10: Ch13 lecture biotechnology

Concept 13.1 Recombinant DNA Can Be Made in the Laboratory

DNA fragments cut by enzymes can be separated by gel electrophoresis.

A mixture of fragments is placed in a well in a semisolid gel, and an electric field is applied across the gel.

Negatively charged DNA fragments move towards the positive end.

Smaller fragments move faster than larger ones.

Page 11: Ch13 lecture biotechnology

Concept 13.1 Recombinant DNA Can Be Made in the Laboratory

DNA fragments separate and give three types of information:

• The number of fragments

• The sizes of the fragments

• The relative abundance of the fragments, indicated by the intensity of the band

Page 12: Ch13 lecture biotechnology

Figure 13.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)

Page 13: Ch13 lecture biotechnology

Figure 13.2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)

Page 14: Ch13 lecture biotechnology

Concept 13.1 Recombinant DNA Can Be Made in the Laboratory

After separation on a gel, a specific DNA sequence can be found with a single-stranded probe.

The gel region can be cut out and the DNA fragment removed.

The purified DNA can be analyzed by sequence or used to make recombinant DNA.

Page 15: Ch13 lecture biotechnology

Concept 13.1 Recombinant DNA Can Be Made in the Laboratory

DNA ligase is an enzyme that catalyzes the joining of DNA fragments, such as Okazaki fragments during replication.

With restriction enzymes to cut fragments and DNA ligase to combine them, new recombinant DNA can be made.

Page 16: Ch13 lecture biotechnology

Figure 13.3 Cutting, Splicing, and Joining DNA

Page 17: Ch13 lecture biotechnology

Concept 13.1 Recombinant DNA Can Be Made in the Laboratory

Recombinant DNA was shown to be a functional carrier of genetic information.

Sequences from two E.coli plasmids, each with different antibiotic resistance genes, were recombined.

The resulting plasmid, when inserted into new cells, gave resistance to both of the antibiotics.

Page 18: Ch13 lecture biotechnology

Figure 13.4 Recombinant DNA (Part 1)

Page 19: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

Recombinant DNA technology can be used to clone (make identical copies) genes.

Transformation: Recombinant DNA is cloned by inserting it into host cells (transfection if host cells are from an animal).

The altered host cell is called transgenic.

Page 20: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

Usually only a few cells exposed to recombinant DNA are actually transformed.

To determine which of the host cells are transgenic, the recombinant DNA includes selectable marker genes, such as genes that confer resistance to antibiotics.

Page 21: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

Most research has been done using model organisms:

• Bacteria, especially E. coli

• Yeasts (Saccharomyces), commonly used as eukaryotic hosts

• Plant cells, able to make stem cells—unspecialized, totipotent cells

• Cultured animal cells, used for expression of human or animal genes—whole transgenic animals can be created

Page 22: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

Methods for inserting the recombinant DNA into a cell:

• Cells may be treated with chemicals to make plasma membranes more permeable—DNA diffuses in.

• Electroporation—a short electric shock creates temporary pores in membranes, and DNA can enter.

Page 23: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

• Viruses and bacteria can be altered to carry recombinant DNA into cells.

• Transgenic animals can be produced by injecting recombinant DNA into the nuclei of fertilized eggs.

• “Gene guns” can “shoot” the host cells with particles of DNA.

Page 24: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

The new DNA must also replicate as the host cell divides.

DNA polymerase does not bind to just any sequence.

The new DNA must become part of a segment with an origin of replication—a replicon or replication unit.

Page 25: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

New DNA can become part of a replicon in two ways:

• Inserted near an origin of replication in host chromosome

• It can be part of a carrier sequence, or vector, that already has an origin of replication

Page 26: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

Plasmids make good vectors:

• Small and easy to manipulate

• Have one or more restriction enzyme recognition sequences that each occur only once

• Many have genes for antibiotic resistance which can be selectable markers

Page 27: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

• Have a bacterial origin of replication (ori) and can replicate independently of the host chromosome

Bacterial cells can contain hundreds of copies of a recombinant plasmid. The power of bacterial transformation to amplify a gene is extraordinary.

Page 28: Ch13 lecture biotechnology

In-Text Art, Ch. 13, p. 249

Page 29: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

A plasmid from the soil bacterium Agrobacterium tumefaciens is used as a vector for plant cells.

A. tumefaciens contains a plasmid called Ti (for tumor-inducing).

The plasmid has a region called T DNA, which inserts copies of itself into chromosomes of infected plants.

Page 30: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

T DNA genes are removed and replaced with foreign DNA.

Altered Ti plasmids transform Agrobacterium cells, then the bacterium cells infect plant cells.

Whole plants can be regenerated from transgenic cells, or germ line cells can be infected.

Page 31: Ch13 lecture biotechnology

In-Text Art, Ch. 13, p. 250

Page 32: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

Most eukaryotic genes are too large to be inserted into a plasmid.

Viruses can be used as vectors—e.g., bacteriophage. The genes that cause host cells to lyse can be cut out and replaced with other DNA.

Because viruses infect cells naturally they offer an advantage over plasmids.

Page 33: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

Usually only a small proportion of host cells take up the vector (1 cell in 10,000) and they may not have the appropriate sequence.

Host cells with the desired sequence must be identifiable.

Selectable markers such as antibiotic resistance genes can be used.

Page 34: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

If a vector carrying genes for resistance to two different antibiotics is used, one antibiotic can select cells carrying the vector.

If the other antibiotic resistance gene is inactivated by the insertion of foreign DNA, then cells with the desired DNA can be identified by their sensitivity to that antibiotic.

Page 35: Ch13 lecture biotechnology

Figure 13.5 Marking Recombinant DNA by Inactivating a Gene

Page 36: Ch13 lecture biotechnology

Concept 13.2 DNA Can Genetically Transform Cells and Organisms

Selectable markers are a type of reporter gene—a gene whose expression is easily observed.

Green fluorescent protein, which normally occurs in a jellyfish, emits visible light when exposed to UV light.

The gene for this protein has been isolated and incorporated into vectors as a reporter gene.

Page 37: Ch13 lecture biotechnology

Figure 13.6 Green Fluorescent Protein as a Reporter

Page 38: Ch13 lecture biotechnology

Concept 13.3 Genes and Gene Expression Can Be Manipulated

DNA fragments used for cloning come from three sources:

• Gene libraries

• Reverse transcription from mRNA

• Products of PCR

• Artificial synthesis or mutation of DNA

Page 39: Ch13 lecture biotechnology

Concept 13.3 Genes and Gene Expression Can Be Manipulated

A genomic library is a collection of DNA fragments that comprise the genome of an organism.

The DNA is cut into fragments by restriction enzymes, and each fragment is inserted into a vector.

A vector is taken up by host cells which produce a colony of recombinant cells.

Page 40: Ch13 lecture biotechnology

Concept 13.3 Genes and Gene Expression Can Be Manipulated

Smaller DNA libraries can be made from complementary DNA (cDNA).

mRNA is extracted from cells, then cDNA is produced by complementary base pairing, catalyzed by reverse transcriptase.

A cDNA library is a “snapshot” of the transcription pattern of the cell.

cDNA libraries are used to compare gene expression in different tissues at different stages of development.

Page 41: Ch13 lecture biotechnology

Figure 13.7 Constructing Libraries

Page 42: Ch13 lecture biotechnology

Concept 13.3 Genes and Gene Expression Can Be Manipulated

DNA can be synthesized by PCR if appropriate primers are available.

The amplified DNA can then be inserted into plasmids to create recombinant DNA and cloned in host cells.

Artificial synthesis of DNA is now fully automated.

Page 43: Ch13 lecture biotechnology

Concept 13.3 Genes and Gene Expression Can Be Manipulated

Synthetic oligonucleotides are used as primers in PCR reactions.

Primers can create new sequences to create mutations in a recombinant gene.

Longer synthetic sequences can be used to construct an artificial gene.

Page 44: Ch13 lecture biotechnology

Concept 13.3 Genes and Gene Expression Can Be Manipulated

Synthetic DNA can be manipulated to create specific mutations in order to study the consequences of the mutation.

Mutagenesis techniques have revealed many cause-and-effect relationships (e.g., determining signal sequences).

Page 45: Ch13 lecture biotechnology

Concept 13.3 Genes and Gene Expression Can Be Manipulated

A knockout experiment inactivates a gene so that it is not transcribed and translated into a functional protein.

In mice, homologous recombination targets a specific gene.

The normal allele of a gene is inserted into a plasmid—restriction enzymes are used to insert a reporter gene into the normal gene.

The extra DNA prevents functional mRNA from being made.

Page 46: Ch13 lecture biotechnology

Concept 13.3 Genes and Gene Expression Can Be Manipulated

The recombinant plasmid is used to transfect mouse embryonic stem cells.

Stem cells—unspecialized cells that divide and differentiate into specialized cells

The original gene sequences line up with their homologous sequences on the mouse chromosome.

Page 47: Ch13 lecture biotechnology

Concept 13.3 Genes and Gene Expression Can Be Manipulated

The transfected stem cell is then transplanted into an early mouse embryo.

The knockout technique has been important in determining gene functions and studying human genetic diseases.

Many diseases have a knockout mouse model.

Page 48: Ch13 lecture biotechnology

Figure 13.8 Making a Knockout Mouse

Page 49: Ch13 lecture biotechnology

Concept 13.3 Genes and Gene Expression Can Be Manipulated

Complementary RNA:

Translation of mRNA can be blocked by complementary microRNAs—antisense RNA.

Antisense RNA can be synthesized and added to cells to prevent translation—the effects of the missing protein can then be determined.

Page 50: Ch13 lecture biotechnology

Concept 13.3 Genes and Gene Expression Can Be Manipulated

RNA interference (RNAi) is a rare natural mechanism that blocks translation.

RNAi occurs via the action of small interfering RNAs (siRNAs).

An sRNA is a short, double stranded RNA that is unwound to single strands by a protein complex, which also catalyzes the breakdown of the mRNA.

Small interfering RNA (siRNA) can be synthesized in the laboratory.

Page 51: Ch13 lecture biotechnology

Figure 13.9 Using Antisense RNA and siRNA to Block the Translation of mRNA

Page 52: Ch13 lecture biotechnology

Concept 13.3 Genes and Gene Expression Can Be Manipulated

DNA microarray technology provides a large array of sequences for hybridization experiments.

A series of DNA sequences are attached to a glass slide in a precise order.

The slide has microscopic wells, each containing thousands of copies of sequences up to 20 nucleotides long.

Page 53: Ch13 lecture biotechnology

Concept 13.3 Genes and Gene Expression Can Be Manipulated

DNA microarrays can be used to identify specific single nucleotide polymorphisms or other mutations.

Microarrays can be used to examine gene expression patterns in different tissues in different conditions.

Example: Women with a propensity for breast cancer tumors to recur have a gene expression signature.

Page 54: Ch13 lecture biotechnology

Figure 13.10 Using DNA Microarrays for Clinical Decision-Making

Page 55: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Almost any gene can be inserted into bacteria or yeasts and the resulting cells induced to make large quantities of a product.

Requires specialized expression vectors with extra sequences needed for the transgene to be expressed in the host cell.

Page 56: Ch13 lecture biotechnology

Figure 13.11 A Transgenic Cell Can Produce Large Amounts of the Transgene’s Protein Product

Page 57: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Expression vectors may also have:

• Inducible promoters that respond to a specific signal

• Tissue-specific promoters, expressed only in certain tissues at certain times

• Signal sequences—e.g., a signal to secrete the product to the extracellular medium

Page 58: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Many medically useful products are being made using biotechnology.

The two insulin polypeptides are synthesized separately along with the β-galactosidase gene.

After synthesis the polypeptides are cleaved, and the two insulin peptides combined to make a functional human insulin molecule.

Page 59: Ch13 lecture biotechnology

Figure 13.12 Human Insulin: From Gene to Drug (Part 1)

Page 60: Ch13 lecture biotechnology

Figure 13.12 Human Insulin: From Gene to Drug (Part 2)

Page 61: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Before giving it to humans, scientists had to be sure of its effectiveness:

• Same size as human insulin

• Same amino acid sequence

• Same shape

• Binds to the insulin receptor on cells and stimulates glucose uptake

Page 62: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Pharming: Production of pharmaceuticals in farm animals or plants.

Example: Transgenes are inserted next to the promoter for lactoglobulin—a protein in milk. The transgenic animal then produces large quantities of the protein in its milk.

Page 63: Ch13 lecture biotechnology

Figure 13.13 Pharming

Page 64: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Human growth hormone (for children suffering deficiencies) can now be produced by transgenic cows.

Only 15 such cows are needed to supply all the children in the world suffering from this type of dwarfism.

Page 65: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Through cultivation and selective breeding, humans have been altering the traits of plants and animals for thousands of years.

Recombinant DNA technology has several advantages:

• Specific genes can be targeted

• Any gene can be introduced into any other organism

• New organisms can be generated quickly

Page 66: Ch13 lecture biotechnology

Figure 13.14 Genetic Modification of Plants versus Conventional Plant Breeding (Part 1)

Page 67: Ch13 lecture biotechnology

Figure 13.14 Genetic Modification of Plants versus Conventional Plant Breeding (Part 2)

Page 68: Ch13 lecture biotechnology

Table 13.2 Potential Agricultural Applications of Biotechnology

Page 69: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Crop plants have been modified to produce their own insecticides:

• The bacterium Bacillus thuringiensis produces a protein that kills insect larvae

• Dried preparations of B. thuringiensis are sold as a safe alternative to synthetic insecticides. The toxin is easily biodegradable.

Page 70: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

• Genes for the toxin have been isolated, cloned, and modified, and inserted into plant cells using the Ti plasmid vector

• Transgenic corn, cotton, soybeans, tomatoes, and other crops are being grown. Pesticide use is reduced.

Page 71: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Crops with improved nutritional characteristics:

• Rice does not have β-carotene, but does have a precursor molecule

• Genes for enzymes that synthesize β-carotene from the precursor are taken from daffodils and inserted into rice by the Ti plasmid

Page 72: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

• The transgenic rice is yellow and can supply β-carotene to improve the diets of many people

• β-carotene is converted to vitamin A in the body

Page 73: Ch13 lecture biotechnology

Figure 13.15 Transgenic Rice Rich in β-Carotene

Page 74: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Recombinant DNA is also used to adapt a crop plant to an environment.

Example: Plants that are salt-tolerant.

Genes from a protein that moves sodium ions into the central vacuole were isolated from Arabidopsis thaliana and inserted into tomato plants.

Page 75: Ch13 lecture biotechnology

Figure 13.16 Salt-tolerant Tomato Plants (Part 1)

Page 76: Ch13 lecture biotechnology

Figure 13.16 Salt-tolerant Tomato Plants (Part 2)

Page 77: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Instead of manipulating the environment to suit the plant, biotechnology may allow us to adapt the plant to the environment.

Some of the negative effects of agriculture, such as water pollution, could be reduced.

Page 78: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Concerns over biotechnology:

• Genetic manipulation is an unnatural interference in nature

• Genetically altered foods are unsafe to eat

• Genetically altered crop plants are dangerous to the environment

Page 79: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Advocates of biotechnology point out that all crop plants have been manipulated by humans.

Advocates say that since only single genes for plant function are inserted into crop plants, they are still safe for human consumption.

Genes that affect human nutrition may raise more concerns.

Page 80: Ch13 lecture biotechnology

Concept 13.4 Biotechnology Has Wide Applications

Concern over environmental effects centers on escape of transgenes into wild populations:

• For example, if the gene for herbicide resistance made its way into the weed plants

• Beneficial insects can also be killed from eating plants with B. thuringiensis genes

Page 81: Ch13 lecture biotechnology

Answer to Opening Question

Bioremediation is the use, by humans, of organisms to remove contaminants from the environment.

Composting and wastewater treatment use bacteria to break down large molecules, human wastes, paper, and household chemicals.

Recombinant DNA technology has transformed bacteria to help clean up oil spills.

Page 82: Ch13 lecture biotechnology

Figure 13.17 The Spoils of War