ure 14 Lect $$$

- some of what Gene/cs and Gene/c Engineering amounts to

INOSITOL Scyllo-inositol is rare and expensive. One of the best ways to prepare this cyclitol depends on an enzymic synthesis (Acetobacter suboxydans) of the 2,4,6/3,5-pentahydroxycyclohexanone (meso-2- inosose), described by Kluyver[1] and starNng from myo-inositol (Husson et al., Carb Res 1998) Inositol dehydrogenase (EC 1.1.1.18) from Bacillus sub5lis catalyzes the oxidaNon of myo-inositol to scyllo-inosose by transfer of the equatorial hydride of the substrate to NAD+ (Daniellou et al., Can J Chem 2006) CORN SYRUP Example: Eight billion pounds per year of high-fructose corn syrup is being commercially produced using immobilized enzymes. [US Patent 4442216. Harvey, 2004.] separaNon of enzyme from cell tic a`achment of enzyme gene g glass or ceramic beads or polymer entrapment in starch or silica gel

rin : alte ing ineer oratory g tic en the lab Gene ial in r mate

BIO 2200, Microbiology, Spring/Summer 2011

Chapter 12, Genetic Engineering June 29, 2011 -Harrison

Advances in Sequencing

[Metzker Nat Rev Gene5cs 2010]

Structure of interac0ons

Biomolecular structure

[Aloy and Russell, Nat Rev Mol Cell Biol, 2006]

Cellular structure

Advances in Knowledge of Structure

 Advances in Func/on

[Searls, Nature Reviews, 2005]

Types of diseases addressed by gene therapy Cancer diseases - 871 trials (66.5%) Vascular diseases - 119 trials (9.1%) Monogenic diseases - 109 trials (8.3%) Infectious diseases - 85 trials (6.5%) Neurological diseases - 20 trials (1.5%) Ocular diseases - 12 trials (0.9%) Healthy volunteers - 22 trials (1.7%)

[Edelstein et al., 2007]

Example of Ex Vivo Gene Therapy: Ashantis ADA treatment

http://commons.wikimedia.org/wiki/ Image:Blood_Donation_12-07-06_1.JPG

1 Acquire

blood from SCID patient, then culture T cells

4 Infusion (11 times) PA317/LASN retrovirus 3 2

Genetically deactivated retrovirus PA317 (murine oncornavirus)

bacterium with plasmid containing normal ADA DNA and a neomycin phosphotransferase gene for selection

Disabling Infectiousness of Retroviruses

Retrovirus LTR promoter is disabled to prevent subsequent transcriptional expression from infected host cell DNA

Retrovirus gag, pol and env proteins are replaced by the articially inserted[http://commons.wikimedia.org/wiki/Image:Retroviruses_production.jpg] from other sources gene and protein products are generated (not the retrovirus RNA/DNA)

Gene$c engineering: altering geneNc material in the laboratory

Bioprospec$ng: the collecNon of biological material and the analysis of its material properNes, or its molecular, biochemical or geneNc content, for the purpose of developing a commercial product. [h4p://
www.med.govt.nz/]

[h#p://www.forbes.com/2000/05/29/feat.html]

Amplifying DNA: The Polymerase Chain Reac/on

Polymerase chain reac5on (PCR): method that produces mulNple copies of DNA in vitro
PCR can amplify a target DNA fragment 1,000,000,000-fold from a small amount of template Uses DNA polymerase Conceived by Kary Mullis

PCR is performed in a thermocycler Three steps: denatura5on, annealing, extension Process takes only a few hours

The Polymerase Chain ReacNon (PCR)

Figure 12.11

Amplifying DNA: The Polymerase Chain ReacNon Thermostable DNA polymerase is used (Taq polymerase)
Isolated from thermophilic bacterium Thermus aqua5cus Stable at 90 degrees Celsius No proofreading ac0vity

Pfu polymerase isolated from Pyrococcus furiosus

More thermostable than Taq Has proofreading ac0vity

Amplifying DNA: The Polymerase Chain Reac/on RT-PCR (Reverse transcriptase-PCR) Reverse transcriptase is used to convert RNA into DNA PCR is then performed on cDNA qPCR (quan5ta5ve PCR) Allows researcher to determine the iniNal number of target genes in a sample

Gene/c Engineering: QuesNons of informaNon management with respect to nucleoNde sequence! How to cut nucleic acid polymers? How to analyze the lengths of nucleic acid polymers? How to read nucleic acid polymers? How to copy nucleic acid polymers? How to synthesis de novo (new) nucleic acid polymers? How to edit nucleic acid polymers? How to study behavior of altered nucleic acid polymers? How to isolate organisms with altered nucleic acid polymers? Dierences between Eukarya and Bacteria?

I. Tools and Techniques of Genetic Engineering

12.1
Restriction and Modication Enzymes 12.2
Nucleic Acid Hybridization and the Southern Blot 12.3
Essentials of Molecular Cloning 12.4
Plasmids as Cloning Vectors

12.1 Restriction and Modication Enzymes

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

Restriction enzymes: recognize specic DNA sequences and cut DNA at those sites

Widespread among prokaryotes Rare in eukaryotes Protect prokaryotes from hostile foreign DNA (e.g., viral genomes) Essential for in vitro DNA manipulation

Three Classes of Restriction Enzymes

Type II cleave DNA within their recognition sequence and are most useful for specic DNA manipulation

Restriction enzymes recognize inverted repeat sequences (palindromes)

Typically 48 base pairs long; EcoRI recognizes a 6 base- pair sequence

Sticky ends or blunt ends

Restriction and Modication of DNA

Figure 12.1

Restriction enzymes protect cell from invasion from foreign DNA

Destroy foreign DNA Must protect their own DNA from inadvertent destruction

Modication enzymes: protect cells DNA for restriction enzymes

Chemically modify nucleotides in restriction recognition sequence Modication generally consists of methylation of DNA

Gel electrophoresis: separates DNA molecules based on size

Electrophoresis uses an electrical eld to separate charged molecules Gels are usually made of agarose, a polysaccharide Nucleic acids migrate through gel toward the positive electrode due to their negatively charged phosphate groups

Gels can be stained with ethidium bromide and DNA can be visualized under UV light

The same DNA that has been cut with different restriction enzymes will have different banding patterns on an agarose gel Size of fragments can be determined by comparison to a standard Restriction map: a map of the location of restriction enzyme cuts on a segment of DNA

Figure 12.2

Optical Mapping of Restriction Fragments

Figure 12.3

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

Southern Blot: a hybridization procedure where DNA is in the gel and probe is RNA or DNA

Northern Blot: RNA is in the gel

 Molecular cloning: isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated Three main steps of gene cloning
1) Isolation and fragmentation of source DNA 2) Inserting DNA fragment into cloning vector 3) Introduction of cloned DNA into host organism

1) Isolation and fragmentation of source DNA

Source DNA can be genomic DNA, RNA, or PCR amplied fragments

Genomic DNA must rst be restriction digested

2) Inserting DNA fragment into cloning vector

Most vectors are derived from plasmids or viruses DNA is generally inserted in vitro DNA ligase: enzyme that joins two DNA molecules

Works with sticky or blunt ends

3) Introduction of cloned DNA into host organism

Transformation is often used to get recombinant DNA into host Some cells will contain desired cloned gene, while other cells will have other cloned genes

Gene library: mixture of cells containing a variety of genes

Shotgun cloning: gene libraries made by cloning random genome fragments

12.4 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 carried out by chemical transformation or electroporation

12.4 Plasmids as Cloning Vectors

pUC19 is a common cloning vector

Modied ColE1 plasmid

Contains ampicillin resistance and lacZ gene Contains polylinker (multiple cloning site) within lacZ gene

Cloning Vector Plasmid pUC19

Figure 12.6

Blue/ White Screening

Blue colonies do not have vector with foreign DNA inserted White colonies have foreign DNA inserted

Insertional activation: lacZ gene is inactivated by insertion of foreign DNA

Inactivated lacZ cannot process Xgal; blue color does not develop

II. Sequencing, Synthesis, and Amplication of DNA

12.5
Sequencing DNA 12.6
Sequencing and Annotating Entire Genomes 12.7
Synthesizing DNA 12.8
Amplifying DNA: The Polymerase Chain Reaction

12.5 Sequencing DNA

Sequencing: determining the precise order of nucleotides in a DNA or RNA molecule Sanger dideoxy method

Invented by Nobel prizewinner Fred Sanger Dideoxy analogs of dNTPs used in conjunction with dNTPs Analog prevents further extension of DNA chain Bases are labeled with radioactivity Gel electrophoresis is then performed on products

Dideoxynucleotides and Sanger Sequencing

Sequencing: determining the precise order of nucleotides in a DNA or RNA molecule Sanger dideoxy method

Invented by Nobel prizewinner Fred Sanger Dideoxy analogs of dNTPs used in conjunction with dNTPs Analog prevents further extension of DNA chain Bases are labeled with radioactivity Gel electrophoresis is then performed on products

Figure 12.8

12.5 Sequencing DNA

Large-scale sequencing projects have led to automated DNA sequencing systems

Based on Sanger method Radioactivity replaced by uorescent dye

454 sequencing system

Recent technological advance Generates data 100 X faster than Sanger method

454 relies on two major advances

Massively parallel liquid handling and pyrosequencing

Light is released each time a base is added to DNA strand Instrument actually measures release of light Can only handle short stretches of DNA

Virtually all genomic sequencing projects use shotgun sequencing

Entire genome is cloned and resultant clones are sequenced Much of the sequencing is redundant Generally 7 to 10-fold coverage

Computer algorithms used to look for replicate sequences and assemble them

Occasionally assembly is not possible Closure can be pursued using PCR to target areas of the genome Closed vs. Draft genome

Closed genome relies on manpower More expensive More information

Annotation: converting raw sequence data into a list of genes present in the genome Great majority of genes encode proteins Functional ORF: an open reading frame that encodes a protein

Computer algorithms used to search for ORFs

Look for start/ stop codons and Shine-Dalgarno sequences

ORFs can be compared to ORFs in other genomes

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, probes, and in site-directed mutagenesis

III. Bacterial Gene Manipulation

12.9
Molecular Methods for Mutagenesis

12.10 Gene Fusions and Reporter Genes

12.9 Molecular Methods for

Conventional mutagens produce mutations at random Site-directed mutagenesis: performed in vitro and introduces mutations at a precise location

Can be used to assess the activity of specic amino acids in a protein Structural biologists have gained signicant insight using this tool

Cassette mutagenesis and knockout mutations

Site-Directed Mutagenesis Using Synthetic DNA

Figure 12.12

Gene Disruption by Cassette Mutagenesis

Figure 12.13

12.10 Gene Fusions and Reporter Genes

Reporter genes

Encode proteins that are easy to detect and assay

e.g., lacZ, luciferase, GFP

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

Green Fluorescent Protein (GFP)

Figure 12.14

Construction and Use of Gene Fusions

Figure 12.15

IV. Advanced Cloning Techniques

12.11 Hosts for Cloning Vectors 12.12 Finding the Right Clone 12.13 Shuttle Vectors and Expression Vectors 12.14 Bacteriophage Lambda as a Cloning Vector 12.15 Vectors for Genomic Cloning and Sequencing

12.11 Hosts for Cloning Vectors

Ideal hosts should be

Capable of rapid growth in inexpensive medium Non-pathogenic Capable of incorporating DNA Genetically stable in culture Equipped with appropriate enzymes to allow replication of the vector

Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae

Hosts for Molecular Cloning

Figure 12.16

12.11 Hosts for Cloning Vectors

Introduction of DNA into mammalian cells is called transfection

Originally done through phagocystis of DNA by host cell Can also be done using microinjection Electroporation Gene gun

DNA Gun for Transfection of

Figure 12.17

Essential to detect the host that has the correct clone Initial screen: antibiotic resistance, plaque formation

Often sufcient for cloning of PCR-generated DNA sequences

If working with a heterogeneous gene library you may need to look more closely If cells express the foreign gene and its expression can be detected then screening is relatively easy May need to look for the DNA if gene not expressed

Antibodies

Blood serum proteins produced by animals Made by injecting animal with specic protein antigen Look for binding of labeled nucleic acid probe to DNA from specic colonies

Nucleic acid probes

12.13 Shuttle Vectors and Expression Vectors

Shuttle vectors: vectors that are stably maintained in two or more unrelated host organisms (i.e., E. coli and B. subtilis or yeast) Bacterial plasmid engineered to function in eukaryotes

Add a eukaryotic origin of replication Add a centromere recognition sequence

12.13 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

lac, trp, tac, trc, lambda PL

Effective transcription terminators are used to prevent expression of other genes on the plasmid

Genetic Map of the Expression Vector pSE420

Figure 12.20

The T7 Expression System

Figure 12.21

12.13 Shuttle Vectors and Expression Vectors

mRNA produced must be efciently translated and there are problems with this always happening

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

12.13 Shuttle Vectors and Expression Vectors

Vectors exist for cloning in eukaryotic cells

Yeast articial chromosomes (YACs) DNA virus SV40 Adenovirus, vaccinia virus, baculovirus

Integrating vectors
Integrate into host chromosome Stably maintained in cell

12.14 Bacteriophage Lambda as a Cloning Vector

Lambda makes a good cloning vector

Well-understood biology Can hold larger amounts of DNA than most plasmids DNA can be efciently packaged in vitro Can efciently infect suitable host particles

Replacement vectors are useful in cloning large DNA fragments

Lambda Cloning Vectors

Figure 12.22

Cosmids: plasmid vectors containing the cos site from the lambda genome

Can be packaged into lambda virions Inserts as large as 50 kilobases accepted Avoids necessity of transforming E. coli Phage particles are more stable than plasmids

12.15 Vectors for Genomic Cloning and Sequencing

Specialized vectors for genome analysis exist

Bacteriophage M13 Bacterial articial chromosomes (BACs) Yeast articial chromosomes (YACs)

Bacteriophage M13 vectors

Clone DNA up to 5 kilobases Contains lacZ for blue/white screening

Figure 12.24a

Cloning Using Bacteriophage M13

Figure 12.24b

BACs

Constructed from the F plasmid Host for a BAC is generally a mutant strain of E. coli

YACs

Can accommodate 200800 kilobases of cloned DNA Replicate like normal yeast chromosomes