PowerPoint ® Lecture Presentations prepared by John Zamora Middle Tennessee State University C H A P T E R © 2015 Pearson Education, Inc. Genetic Engineering and Biotechnology 11

© 2015 Pearson Education, Inc Restriction Enzymes and Nucleic Acid Separation Genetic engineering: using in vitro techniques to alter genetic material in the laboratory Basic techniques include: Restriction enzymes Gel electrophoresis Nucleic acid hybridization Nucleic acid probes Molecular cloning Cloning vectors

© 2015 Pearson Education, Inc Restriction Enzymes and Nucleic Acid Separation Restriction enzymes: recognize specific DNA sequences and cut DNA at those sites Widespread among prokaryotes Defense against foreign DNA Rare in eukaryotes Essential molecular tools for in vitro DNA manipulation

© 2015 Pearson Education, Inc Restriction Enzymes and Nucleic Acid Separation Three classes of restriction enzymes Type II cleave DNA within their recognition sequence and are most useful for specific DNA manipulation Restriction enzymes recognize palindromes (inverted repeat sequences) Typically 4–8 base pairs long Sticky ends or blunt ends RACECAR EYE MADAM

© 2015 Pearson Education, Inc. Figure 11.1a 11.1 Restriction Enzymes and Nucleic Acid Separation

© 2015 Pearson Education, Inc Restriction Enzymes and Nucleic Acid Separation Modification enzymes: protect cell's DNA for restriction enzymes Chemically modify nucleotides in restriction recognition sequence Modification generally consists of methylation of DNA Figure 11.1b

© 2015 Pearson Education, Inc Restriction Enzymes and Nucleic Acid Separation Gel electrophoresis: separates DNA molecules based on size Electrophoresis uses an electrical field to separate charged molecules Gels are usually made of agarose, a polysaccharide Nucleic acids migrate through gel toward the positive electrode because of their negatively charged phosphate groups Gels can be stained (e.g. ethidium bromide) so DNA can be visualized under UV light

© 2015 Pearson Education, Inc. Figure 11.2a 11.1 Restriction Enzymes and Nucleic Acid Separation Figure 11.2b

© 2015 Pearson Education, Inc Restriction Enzymes and Nucleic Acid Separation The same DNA that has been cut with different restriction enzymes will result in different banding patterns on an agarose gel Size of fragments can be determined by comparison to a standard called a DNA ladder Figure 11.2b

© 2015 Pearson Education, Inc Nucleic Acid Hybridization Nucleic acid hybridization: base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix Segment of single-stranded DNA that is used in hybridization and has a predetermined identity is called a nucleic acid probe Figure 11.3 Southern blot (DNA) Northern blot (DNA)

© 2015 Pearson Education, Inc Nucleic Acid Hybridization FISH: Fluorescent In Situ Hybridization Uses fluorescent probe attached to oligonucleotide Figure 11.4

© 2015 Pearson Education, Inc The Polymerase Chain Reaction The polymerase chain reaction (PCR) is basically DNA replication in a test tube Kary Mullis

© 2015 Pearson Education, Inc The Polymerase Chain Reaction The polymerase chain reaction (PCR) is basically DNA replication in a test tube Also called DNA amplification, product = amplicon Figure 11.5

© 2015 Pearson Education, Inc The Polymerase Chain Reaction Variations of PCR Reverse transcription PCR (RT PCR) (Fig. 11.6) Can make DNA from an RNA template Uses the enzyme reverse transcriptase Quantitative PCR (q PCR) Uses fluorescent probe to monitor the amplification process

© 2015 Pearson Education, Inc Essentials of Molecular Cloning Molecular cloning: isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated Figure 11.7

© 2015 Pearson Education, Inc Essentials of Molecular Cloning Essential to detect the correct clone Initial screen: antibiotic resistance, plaque formation If cells express the foreign gene and its expression can be detected, then screening is relatively easy Nucleic acid probes/PCR – if gene is not expressed Antibodies – if gene is expressed Figure 11.8

© 2015 Pearson Education, Inc Molecular Methods for Mutagenesis Synthetic DNA Systems are available for de novo synthesis of DNA Oligonucleotides of 100 bases can be made Multiple oligonucleotides can be ligated together Synthesized DNA is used for primers and probes, and in site-directed mutagenesis George Church

© 2015 Pearson Education, Inc Molecular Methods for Mutagenesis Site-directed mutagenesis: performed in vitro and introduces mutations at a precise location Can be used to assess the activity of specific amino acids in a protein Structural biologists have gained significant insight using this tool Figure 11.9 Michael Smith Nobel Prize1993

© 2015 Pearson Education, Inc Molecular Methods for Mutagenesis Cassette mutagenesis and knockout mutations DNA fragment can be cut, excised, and replaced by a synthetic DNA fragment (DNA cassettes or cartridges) The process is known as cassette mutagenesis Gene disruption occurs when cassettes are inserted into the middle of the gene = knockout mutations Figure 11.10

© 2015 Pearson Education, Inc Gene Fusions and Reporter Genes Reporter genes Encode proteins that are easy to detect and assay Examples: lacZ, luciferase, GFP genes Figure 11.11

© 2015 Pearson Education, Inc Gene Fusions and Reporter Genes Gene fusions Promoters or coding sequences of genes of interest can be swapped with those of reporter genes to elucidate gene regulation under various conditions Figure 11.12

© 2015 Pearson Education, Inc Plasmids as Cloning Vectors Plasmids are natural vectors and have useful properties as cloning vectors Small size; easy to isolate DNA Independent origin of replication Multiple copy number; get multiple copies of cloned gene per cell Presence of selectable markers Vector transfer is carried out by chemical transformation or electroporation

© 2015 Pearson Education, Inc Plasmids as Cloning Vectors pUC19 is a common cloning vector Modified ColE1 plasmid Contains ampicillin-resistance Contains multiple cloning site within lacZ gene

© 2015 Pearson Education, Inc. Figure Plasmids as Cloning Vectors

© 2015 Pearson Education, Inc Plasmids as Cloning Vectors Blue/white screening Blue colonies do not have vector with foreign DNA inserted White colonies have foreign DNA inserted Insertional inactivation: lacZ gene is inactivated by insertion of foreign DNA Inactivated lacZ cannot process Xgal; blue color does not develop Figure 11.14

© 2015 Pearson Education, Inc Plasmids as Cloning Vectors Other plasmid vectors Some vectors developed for cloning DNA products made by PCR Some vectors select for recombinant DNA using viability Figure 11.15

© 2015 Pearson Education, Inc Hosts for Cloning Vectors Ideal hosts should be: Capable of rapid growth in inexpensive medium Nonpathogenic Capable of incorporating DNA Genetically stable in culture Equipped with appropriate enzymes to allow replication of the vector Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, others?

© 2015 Pearson Education, Inc. Figure Hosts for Cloning Vectors

© 2015 Pearson Education, Inc Shuttle Vectors and Expression Vectors Shuttle vectors: vectors that are stably maintained in two or more unrelated host organisms (e.g., E. coli and B. subtilis or E. coli and yeast) Bacterial plasmid engineered to function in eukaryotes Add a eukaryotic origin of replication Add a centromere recognition sequence

© 2015 Pearson Education, Inc. Figure Ampicillin resistance oriY oriC t/pa ESM Promoter CEN MCS t/pa E. coli and S. cerevisiae

© 2015 Pearson Education, Inc Shuttle Vectors and Expression Vectors Expression vectors: allow experimenter to control the expression of cloned genes Based on transcriptional control Allow for high levels of protein expression Strong promoters Efficient operators Effective transcription terminators are used to prevent expression of other genes on the plasmid

© 2015 Pearson Education, Inc Shuttle Vectors and Expression Vectors In T7 expression vectors, cloned genes are placed under control of the T7 promoter = hyperexpression Gene for T7 RNA polymerase present and under control of easily regulated system (e.g., lac) T7 RNA polymerase recognizes only T7 promoters Transcribes only cloned genes Shuts down host transcription

© 2015 Pearson Education, Inc. Figure Shuttle Vectors and Expression Vectors

© 2015 Pearson Education, Inc Shuttle Vectors and Expression Vectors mRNA produced must be efficiently translated; problems with this always occur Bacterial ribosome-binding sites are not present in eukaryotic genomes Differences in codon usage between organisms Eukaryotic genes containing introns will not be expressed properly in prokaryotes

© 2015 Pearson Education, Inc Other Cloning Vectors Bacteriophage lambda Modified lambda makes a good cloning vector Well-understood biology Can hold larger amounts of DNA than most plasmids DNA can be efficiently packaged in vitro Can efficiently infect suitable host particles Lambda vectors are useful in cloning large DNA fragments

© 2015 Pearson Education, Inc Other Cloning Vectors Cloning with lambda 1.Isolate vector DNA from phage particles, and cut it with the appropriate restriction enzyme 2.Connec the lambda fragments to foreign DNA using DNA ligase 3.Package the DNA by adding cell extracts containing the head and tail proteins 4.Infect E. coli cells, and isolate phage clones by picking plaques 5.Check the recombinant phage for the presence of foreign DNA Figure 11.20a

© 2015 Pearson Education, Inc Other Cloning Vectors Cosmids: plasmid vectors containing the cos site from the lambda genome Can be packaged into lambda virions Inserts as large as 50 kilobases are accepted Phage particles are more stable than plasmids Specialized vectors for genome analysis exist Bacterial artificial chromosomes (BACs): Constructed from the F plasmid Yeast artificial chromosomes (YACs)

© 2015 Pearson Education, Inc Vectors for Genomic Cloning and Sequencing Yeast artificial chromosomes (YACs) Can accommodate 200–800 kilobases of cloned DNA Replicate like normal yeast chromosomes Figure 11.22

© 2015 Pearson Education, Inc Expressing Mammalian Genes in Bacteria Biotechnology Use of living organisms for industrial or commercial applications Genetically modified organism (GMO) Organism whose genome has been altered

© 2015 Pearson Education, Inc Somatotropin and Other Mammalian Proteins Insulin was the first human protein made commercially by genetic engineering Somatotropin, a growth hormone, is another widely produced hormone Cloned as cDNA from the mRNA Recombinant bovine somatotropin (rBST) is commonly used in the dairy industry; stimulates milk production in cows

© 2015 Pearson Education, Inc. Figure Somatotropin and Other Mammalian Proteins

© 2015 Pearson Education, Inc Transgenic Organisms in Agriculture and Aquaculture Transgenic organism Organism that contains a gene from another organism Plants can be genetically modified through several approaches, including: Electroporation, Particle gun methods, plasmids from bacterium Agrobacterium tumefaciens Many successes in plant genetic engineering; several transgenic plants are in agricultural production

© 2015 Pearson Education, Inc. Figure Transgenic Organisms in Agriculture and Aquaculture

© 2015 Pearson Education, Inc Transgenic Organisms in Agriculture and Aquaculture The plant pathogen Agrobacterium tumefaciens can be used to introduce DNA into plants A. tumefaciens contains the Ti plasmid, which is responsible for virulence The Ti plasmid contains genes that mobilize DNA for transfer to the plant The segment of the Ti plasmid that is transferred to the plant is called the T-DNA

© 2015 Pearson Education, Inc. Figure "Disarmed" Ti plasmid Mobilized region Foreign DNA Kanamycin resistance Spectinomycin resistance Cloning vector Origin A. tumefaciens Origin E. coli 1. Transfer to E.coli cells. E. coli 2. Transfer by conjugation. A. tumefaciens Plant cell Nucleus Chromosomes D-Ti 3. Transfer to plant cells. 4. Grow transgenic plants from plant cells Transgenic Organisms in Agriculture and Aquaculture

© 2015 Pearson Education, Inc Transgenic Organisms in Agriculture and Aquaculture Several areas are targeted for genetic improvements in plants, including resistance to herbicides, insects, and microbial disease, as well as improved product quality Plants are engineered to have herbicide resistance to protect them from herbicides applied to kill weeds (e.g. glyphosate)

© 2015 Pearson Education, Inc. Figure Transgenic Organisms in Agriculture and Aquaculture Glyphosate Resistant Glyphosate Sensitive

© 2015 Pearson Education, Inc Transgenic Organisms in Agriculture and Aquaculture One widely used approach for genetically engineering insect resistance in plants involves introducing genes encoding the toxic protein of Bacillus thuringiensis (Bt toxin) Figure Bt -Bt Beet army worm infestation of tobacco

© 2015 Pearson Education, Inc Transgenic Organisms in Agriculture and Aquaculture Genetic engineering can be used to develop transgenic animals Transgenic animals are useful for: Improving livestock and other animals for human consumption Figure 11.31

© 2015 Pearson Education, Inc Genetically Engineered Vaccines Vector vaccine Vaccine made by inserting genes from a pathogenic virus into a relatively harmless carrier virus (e.g., vaccinia virus) Figure 11.32

© 2015 Pearson Education, Inc Genetically Engineered Vaccines Subunit vaccines Contain only a specific protein or proteins from a pathogenic organism (e.g., coat protein of a virus) Preparation of a viral subunit vaccine Fragmentation of viral DNA by restriction enzymes Cloning of viral coat protein genes into a suitable vector

© 2015 Pearson Education, Inc Mining Genomes Gene mining The process of isolating potentially useful novel genes from the environment without culturing the organism Figure 11.33

© 2015 Pearson Education, Inc Engineering Metabolic Pathways Pathway engineering The process of assembling a new or improved biochemical pathway using genes from one or more organisms Figure 11.34

© 2015 Pearson Education, Inc. NEST: Novel Enteric Synbiotic Technology Probiotic = live microorganism that provides a health benefit Prebiotic = substances that induce the growth or activity of microorganisms Synbiotic = Combination of probiotic and prebiotic that provides a synergistic benefit

© 2015 Pearson Education, Inc. NEST: Novel Enteric Synbiotic Technology Our goal is to engineer selective metabolism of a rare carbohydrate into a probiotic In this system – the addition of a selective prebiotic will regulate the proliferation and persistence of the engineered microbe This microbe can then be used to deliver beneficial proteins to the intestine

© 2015 Pearson Education, Inc. Synbiotic Engineering POSITIVE SELECTION ‘Common’ Nutrient ‘Rare’ Nutrient

© 2015 Pearson Education, Inc Synthetic Biology Synthetic biology – using genetic engineering to create novel biological systems out of available parts (biobricks)

© 2015 Pearson Education, Inc Synthetic Biology Synthetic biology – using genetic engineering to create novel biological systems out of available parts (biobricks) Figure 11.35