14th week:

Chapter 11.
Genetic Engineering
(유전공학)
 Chapter 11

Chapter 12
(12th Edition)
Genetic Engineering
(유전공학)
 Genetic Map of the Escherichia coli
Chromosome
• Escherichia coli is a useful model organism for the study of
biochemistry, genetics, and bacterial physiology
• The E. coli chromosome from strain MG1655 has been mapped using
conjugation, transduction, molecular cloning, and sequencing
• Some Features of the E. coli Chromosome
– Many genes encoding enzymes of a single biochemical pathway are clustered
into operons
– Operons equally distributed on both strands
– ~ 5 Mbp in size
– ~ 40% of predicted proteins are of unknown function
– Average protein contains ~ 300 amino acids
– Insertion sequences (IS elements)
Circular Linkage Map of the Chromosome of E. coli K-12

Source: Brock Biology of Microorganisms 12th edition.

Figure 12.1
 Tools and Techniques
• 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 specific 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
 Tools and Techniques
• Three Classes of Restriction Enzymes
– Type II cleave DNA within their recognition sequence and are most useful for
specific DNA manipulation
• Restriction enzymes recognize inverted repeat sequences
(palindromes)
– Typically 4–8 base pairs long; EcoRI recognizes a 6 base- pair sequence
• Sticky ends or blunt ends
• Restriction enzymes protect cell from invasion from foreign DNA
– Destroy foreign DNA
– Must protect their own DNA from inadvertent destruction
• Modification enzymes: protect cell’s DNA for restriction enzymes
– Chemically modify nucleotides in restriction recognition sequence
– Modification generally consists of methylation of DNA
 Tools and Techniques
• 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 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
 Tools and Techniques
• 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
 Tools and Techniques
• 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
• pUC19 is a common cloning vector - Modified ColE1 plasmid
– contains ampicillin resistance and lacZ gene
– Contains polylinker (multiple cloning site) within lacZ gene
• 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
 Sequencing DNA
• Sequencing: determining the precise order of nucleotides in a DNA or
RNA molecule
• Sanger dideoxy method
– Invented by Nobel prize winner 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
• Large-scale sequencing projects have led to automated DNA sequencing systems
– Based on Sanger method
– Radioactivity replaced by fluorescent dye
• 454 sequencing system
– Recent technological advance
– Generates data 100 X faster than Sanger method
 Sequencing DNA
• 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
• Annotation: converting raw sequence data into a list of genes present
in the genome
• Great majority of genes encode proteins
 Sequencing DNA
• 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
• 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
 Synthesizing and Amplifying 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, probes, and in site-directed
mutagenesis

• Polymerase chain reaction (PCR): method that produces multiple

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: denaturation, annealing, extension
• Process takes only a few hours
 Synthesizing and Amplifying DNA
• During each round of PCR the amount of product doubles
– Leads to an exponential increase in DNA
– Only a few molecules of target DNA are needed
– PCR is valuable for cloning and sequencing purposes
– PCR has been used to amplify DNA from mummified remains, fossilized plants
and animals
• Thermostable DNA polymerase is used (Taq polymerase)
– Isolated from thermophilic bacterium Thermus aquaticus
– Stable at 90 degrees Celsius
– No proofreading activity
• RT-PCR (Reverse transcriptase-PCR)
– Reverse transcriptase is used to convert RNA into DNA
– PCR is then performed on cDNA
• qPCR (quantitative PCR)
– Allows researcher to determine the initial number of target genes in a sample
 Bacterial Gene Manipulation
• 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 specific amino acids in a protein
– Structural biologists have gained significant insight using this tool
• Cassette mutagenesis and knockout mutations
• 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
 Advanced Cloning Techniques
• 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
• 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
Hosts for Molecular Cloning

Source: Brock Biology of Microorganisms 12th edition.

Figure 12.16
 Advanced Cloning Techniques
• 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
• 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
• mRNA produced must be efficiently 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
 Genetic Map of a Shuttle Vector Used in Yeast
Transcription termination
/polyadenylation signals

Origin of replication Eukaryotic selectable marker

Yeast centromere sequence

Source: Brock Biology of Microorganisms 12th edition.
Figure 12.19
 Advanced Cloning Techniques
• Vectors exist for cloning in eukaryotic cells
– Yeast artificial chromosomes (YACs)
– DNA virus SV40
– Adenovirus, vaccinia virus, baculovirus
• Integrating vectors
– Integrate into host chromosome
– Stably maintained in cell
• 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
• Replacement vectors are useful in cloning large DNA fragments
 Advanced Cloning Techniques
• 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
• Specialized vectors for genome analysis exist
– Bacteriophage M13
– Bacterial artificial chromosomes (BACs)
– Yeast artificial chromosomes (YACs)
• Bacteriophage M13 vectors
– Clone DNA up to 5 kilobases, Contains lacZ for blue/white screening
• BACs
– Constructed from the F plasmid, Host for a BAC is generally a mutant strain of E. coli
• YACs
– Can accommodate 200–800 kilobases of cloned DNA, Replicate like normal yeast chromosomes