853 MCB 3020, Spring 2005 Chapter 31: Genetic Engineering and Biotechnology I

854 Genetic Engineering I I. Genetic Engineering II. Cloning vectors

855 I. Genetic Engineering i. Some uses ii. Restriction enzymes iii. DNA cloning iv. DNA library

856 Genetic Engineering DNA manipulation using molecular biology techniques DNA cloning identification of genes of interest expression of genes to make a desired product Typical procedures

857 i. Some uses of genetic engineering Industrial or biotechnology products –(eg. alkaliphilic proteases, Taq polymerase) Medical products –(eg. insulin, hepatitis B vaccines, gene therapy) Agriculture and Environment –(eg. plant resistant to pesticides, insects, disease) Basic research –(answers to fundamental questions about life)

858 ii. Restriction enzymes a. natural role b. recognition sequence c. cut sites d. modification enzymes

859 ii. Restriction enzymes Enzymes that break double-stranded DNA at specific sequences. Used to protect bacteria from viruses (by cutting viral DNA) Bacterial DNA is protected by modification enzymes. TB a. Natural role

860 b. Recognition sequence DNA sequence where cutting occurs EcoRI recognition sequence GAATTC CTTAAG cut sites Palindromic recognition sequence TB

861 GAATTC CTTAAG After cutting double stranded break sticky ends TB

862 TTTAAA AAATTT TTT AAA TTT After cutting: blunt ends DraI DraI recognition sequence TB

863 c. Modification enzymes covalently modify DNA, often by methylation protect bacteria from their own restriction enzymes recognize the same site on DNA as the corresponding restriction enzyme prevent cutting by the corresponding restriction enzyme

864 GAATTC CH 3 EcoRI methylase Typical modification enzyme CTTAAG methylation CH 3 TB After modification, the EcoRI restriction enzyme will NOT recognize the methylated DNA.

865 iii.DNA cloning Isolation and insertion of a DNA fragment (insert) into a vector. ori + Cloning vector a small independently replicating genetic element into which genes can be recombined

866 Basic steps of DNA cloning 2. Digest source DNA and vector using restriction enzymes 3. Ligate the source DNA to the vector 4. Introduce DNA into a host 5. Identify the clone of interest TB 1. Isolate source DNA

867 source DNA cloning vector One to many clones representing the source DNA TB 2. Digest DNA and vector cloned DNA (insert) vector 3. Ligate host cell 4. Introduce DNA into host 5. Identify clone of interest 1. Isolate

Isolate source DNA xx eg. Bam HI x source DNA 2. Digest source DNA and vector using restriction enzymes gene of interest

869 restriction site 3. Ligate source DNA into vectors. source DNA ori cloning vector + x + cloned DNA (insert) X

Introduce DNA into a host. + E. coli DNA library Plate on agar X X

Identify the clone of interest Plate on selective medium to find colonies with cloned DNA E. coli with cloned DNA Identify colonies with gene of interest

872 iv. DNA library A large number of clones representing the entire genome of an organism. The source DNA for DNA libraries is typically the genomic DNA. Introduce into host cells; plate X x

873 II. Cloning vectors A. important features B. examples restriction site ori Ap R

means of replication (ori) 2. unique restriction sites (single cut) 3. selectable markers 4. gene inactivation marker A. Important features restriction site ori Ap R Ap R = ampicillin resistance gene Tc R Tc R = tetracycline resistance gene

875 Selectable marker In genetic engineering, a gene whose product can be used to select the cells that carry the plasmid of interest Gene inactivation marker In genetic engineering, a gene that is disrupted (inactivated) when a second gene of interest is cloned into the plasmid or DNA

876 4a. Selectable markers and gene inactivation Uncut vector allows cells to grow on ampicillin (Ap) and tetracycline (Tc). What happens when foreign DNA is inserted into the BamH 1 site? Bam H1 ori Ap R Tc R transform E. coli + ampicillin replica plating + ampicillin + tetracycline the Tc R (tetracyline resistance) gene is inactivated

877 Selectable markers and gene inactivation When foreign DNA is inserted, Tc R gene is inactivated cells will grow on Ap, but NOT tetracycline Bam H1 ori Ap R Tc R Bam H1 digest Inactivated Tc R Ap R Tc R Ap R + Tc R Ap R insert

878 Cells containing the cloned DNA (insert), are Ap-resistant (Ap R ) but Tc-sensitive (Tc S ). replica plating + ampicillin + tetracycline In this gene inactivation system, what happens when E. coli is transformed with a mixture of vector and [vector with insert]? + ampicillin * * * Ap R Tc R Ap R Tc R X mixture of vector and [vector with insert]

879 4b. Another gene inactivation marker is the lacZ gene (codes for beta-galactosidase) Bam H1 ori Ap R lacZ Cl Br N O O X-gal (CLEAR) BLUE product beta-galactosidase cleaves X-gal and produces a blue color Cl Br N HO O OH beta-galactosidase

880 O OH Colonies containing vector WITHOUT an insert are blue. + ampicillin + X-gal (We don't want these.) Bam H1 ori Ap R lacZ

881 When foreign DNA is inserted, it inactivates lacZ beta-galactosidase is not made X-gal is not cleaved colonies with insert are white, NOT blue X X-gal (CLEAR) + ampicillin + X-gal X (LacZ-) insert

882 B. Examples of cloning vectors 1. Plasmids 2. Phage 3. Cosmids 4. YACs

883 pBR322 Bam HI Bam HI = unique restriction site Ap R Ap R = ampicillin resistance gene Tc R Tc R = tetracycline resistance gene ori ori = origin of DNA replication vector 1. plasmid vector (holds ~10 kb) source DNA Bam HI sites

884 Bam HI digestion mixture of [vector with cloned DNA], and vector ligation cloned DNA Tc R source DNA TB

885 a. Phage lambda ( ) dsDNA 1/3 of genome non-essential for lytic growth 2. Phage vector (holds about 20 kb) (can replace this section with foreign DNA) TB

886 e.g. of phage vector: Charon 4A (genetically altered derivative) lacZ gene cos site EcoRI sites 1. restriction 2. ligation cloned DNA TB

Package the cloned DNA into capsids in vitro. 4. Infect host cells and plate to obtain plaques lawn of E. coli cells plaques (regions of dead cells caused by lytic phage)

888 blue plaques (LacZ+) clear plaques (LacZ-) 5. Isolate DNA from clear plaques. (Blue plaques do NOT have insert. We don't want these.) TB

889 b. Phage M13 vectors Phage M13: a ssDNA virus that has a dsDNA replicative form Used to produce ssDNA for DNA sequencing and site- directed mutagenesis Double-stranded replicative form is used for cloning

Cosmid (holds up to 45 kb) Plasmids with cos (cohesive end) sites for in vitro packaging into capsids. plasmid cos 1. clone DNA fragments 2. linearize 3. package in vitro

Yeast Artificial chromosomes (YACs) (holds up to 800 kb) ori telomeres centromere cloning site selectable marker kb inserts (Human genome ~ 3 x 10 9 bp or 3 x 10 6 kb) Features of YACS:

892 Comparison of clone sizes Plasmids up to ~10 kb Charon phage up to ~ 20 kb Cosmids up to ~ 45 kb YACS up to ~800 kb

893 C. Hosts for cloning vectors Escherichia coli Bacillus subtilis Saccharomyces cerevisiae (yeast) mammalian cells

894 Study objectives 1. Name three procedures typically used in genetic engineering. 2. What are some uses of genetic engineering? Know the examples presented. 3. What are restriction and modification enzymes? What is their natural role? Describe the general features of the recognition site of restriction enzymes. You do NOT need to memorize the sequences of the recognition sites. 4. What is DNA cloning? What is a cloning vector? 5. Understand in detail the basic steps involved in cloning DNA. 6. What is a DNA library? What is the typical source DNA for a library? 7. Know the important features of a cloning vector and their roles in cloning. 8. Describe how antibiotic resistance genes and the beta-galactosidase gene can be used to determine if foreign DNA has been inserted into a vector. 9. Understand why the following are important for cloning vectors: selectable markers, gene inactivation, means of replication, unique restriction sites. 10. How the following are used in DNA cloning: plasmid vectors (example, pBR322) phage vectors (examples, Charon 4A and M13) cosmids, and YACs. 11. Compare and contrast the different DNA cloning vectors. What features are specific to each cloning vector? 12. Know that specific host cells facilitate cloning. Know the examples presented.

895 MCB 3020 Spring 2005 Chapter 31: Genetic Engineering and Biotechnology II

896 Last time: I. Genetic Engineering II. Cloning vectors III. Identifying clones of interest IV. Expression vectors V. Polymerase chain reaction (PCR) VI. Cloning and expression of mammalian genes in bacteria VII. Applications of genetic engineering Today:

897 III. Identifying clones of interest A. antibodies B. DNA and RNA probes C. complementation

898 A. antibodies (immunoglobulins) soluble immune system proteins that bind specific antigens* TB (*Antigens are "nonself" (foreign) molecules that interact with components of the immune system.) This antigen is a protein.

899 Using antibodies to identify clones 3. If a DNA clone expresses protein X, it includes the gene for protein X 1. Purify protein of interest (protein X). X Y 2. Prepare antibody ( ) that specifically binds to protein X. Y 4. Use antibody to test clones for production of protein X. TB

900 transformant colonies DNA Library transform E. coli 1. replica plate cells to filter paper transformant cells on filter paper TB Using antibodies to identify clones of interest

lyse cells 3. bind the antibody 4. detect the antibody contains a DNA clone expressing the protein of interest TB

902 B. DNA and RNA probes Probe: labeled DNA or RNA that can bind a particular DNA by complementary base paring. (Probes can be short single-stranded oligonucleotides with a radioactive or fluorescent label attached) 32 P

903 Uses of DNA probes 1. Detect DNA with a sequence related to a DNA of known sequence. 2. Detect genes that encode proteins of partially known sequence. TB

lyse cells 3. bind and detect probe 2. denature DNA contains a clone with sequences complementary to the probe transformant cells on filter paper TB

905 C. Complementation: How could genes of interest be identified by complementation? Restoration of the wildtype phenotype by a second DNA molecule TB X human DNA library mutation X E. coli coenzyme B12 mutant (can't make coenzyme B12) X

906 IV. Expression vectors A. Factors affecting protein expression B. Typical expression vector gene for regulatory protein P O Ap R Vectors used to produce large amounts of protein. ori

907 Expression vectors Vectors used for the production of proteins. usually used to get a high level of gene expression TB

908 A. Factors affecting protein expression 2. Promoter strength and regulation 3. Translation initiation 4. Codon usage 5. Protein and mRNA stability TB 1. Gene copy number

909 B. Typical expression vector P O P = promoter O = operator gene of interest selectable marker unique restriction site ori lacI gene (encodes repressor protein) TB

910 P O lacZ lacY lacA Lactose ( ) induces the expression of lac genes or whatever genes follow the lac promoter. CAP site Some repressor proteins mediate gene induction. + P O gene of interest normal lac operon genetically engineered gene protein of interest

911 V. Polymerase chain reaction (PCR) A. applications B. reaction components C. procedure Process for producing large amounts of DNA from a small amount of template DNA.

912 A. PCR applications gene cloning mutagenesis amplification of related sequences TB amplification of small amounts of DNA for

913 B. PCR reaction components the 4 deoxynucleotides buffer template (~10 4 molecules) thermostable DNA polymerase ( Taq or Pfu polymerase) 2 DNA primers (10 17 molecules) TB

914 The 2 DNA primers bind on opposite strands of DNA 5' Primer #2 Primer #1 Heat to separate strands Cool to anneal to primers primers template TB

denature template DNA template primers DNA polymerase denature at 94°C C. procedure TB

916 anneal at ~ 50ºC 2 anneal primers primers bind by complementary base pairing TB

917 extend at 72ºC 3. extend with DNA polymerase 4. repeat steps 1-3, ~ 35 times (35 cycles) TB

918 denature second cycle TB

919 anneal second cycle TB

920 extend second cycle TB

cycles template final product primers are incorporated into product TB

922 = (number of templates) x 2 (number of cycles) = (1) x 2 35 = 3.4 x molecules Amount of product from 1 molecule 34,000,000,000 TB

923 A. Problems introns large genomes posttranslational modifications (like glycosylation, attaching a sugar) One solution to the intron problem: cDNA ("complementary DNA") VI. Cloning and expression of mammalian genes in bacteria TB

924 B. cDNA mRNA AAAA... TTTT... alkali (removes mRNA) reverse AAAA... TTTT... transcriptase primer TB

925 DNA polymerase clone specific nuclease cDNA TB

926 VII. Applications of genetic engineering A. General uses B. Mammalian proteins C. Vaccines D. Plants E. Gene transfer to plants by bacteria TB

927 A. General uses Microbial fermentations (eg. antibiotic production) Vaccines Mammalian proteins TB Transgenic plants and animals Environmental biotechnology Gene therapy

928 B. Mammalian proteins Insulin alpha-interferon clotting factors TB C. Vaccines Hepatitis B

929 D. Genetic engineering in plants Disease resistance Improved product quality Production of pharmaceuticals TB Herbicide resistance Insect resistance

930 cloned DNA transfer sequences E. Gene transfer to plants by bacteria plasmid used for gene transfer Kan R Kan R = kanamycin resistance TB

931 Plant cell genome Agrobacterium tumefaciens transgenic plant regeneration D-Ti Provides genes needed for DNA Transfer TB

932 Study objectives 1. Understand the details of how antibodies, nucleic acid probes and complementation are used to identify particular clones. 2. Know the main factors that affect protein expression from expression vectors. 3. Understand the polymerase chain reaction, its uses, and the details of the procedure presented in class. 4. Understand how cDNA is made and how it solves some of the problems of cloning eukaryotic genes. 5. What are some of the applications of genetic engineering? 6. Understand how Agrobacterium can be used to transfer genes to plants.