gene cloning
TRANSCRIPT
What Does It Mean: “To Clone”?What Does It Mean: “To Clone”?
Clone: a collection of molecules or cells, all identical to an original molecule or cell
To "clone a gene" is to make many copies of it - for example, by
replicating it in a culture of bacteria.Cloned gene can be a normal copy of a gene (= “wild type”).Cloned gene can be an altered version of a gene (= “mutant”).Recombinant DNA technology makes manipulating genes
possible.
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One basic cloning technique begins with the insertion of a foreign gene into a bacterial plasmid.
02/20/15Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
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The objectives of Recombinant DNA technology include: ◦ Identifying genes◦ Isolating genes◦ Modifying genes◦ Re-expressing genes in other hosts or
organisms
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Genetic engineering produces proteins that offer advantages over proteins isolated from other biological sources. These advantages include:◦ High purity
◦ High specific activity
◦ Steady supply
◦ Batch-to-batch consistency
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Case Study: The Use of Recombinant Case Study: The Use of Recombinant DNA to Produce Human InsulinDNA to Produce Human Insulin
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Why synthesize human insulin?Why synthesize human insulin?Patients’ immune systems do not
produce antibodies against human insulin as they do with bovine or porcine insulin
Projected decline in the production of animal-derived insulin
Need for a more reliable and sustainable method of obtaining the product
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Why is insulin needed?Why is insulin needed?Protein hormone produced by beta cells
of islets of Langerhans in the pancreasRegulates blood sugar by allowing uptake
of glucose from bloodstream into body cells
Patients with diabetes have insufficient or impaired production of insulin
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Structure of InsulinStructure of InsulinTwo polypeptide chains; one with 21
amino acids and the second with 30 amino acids
Chains are linked via a disulfide bond
Gene encoding the insulin protein is found on chromosome 11
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Recombinant DNA TechniqueRecombinant DNA Technique
Restriction enzymes used to cut out insulin gene and to cut a bacterial
(E. coli) plasmid at the same “sticky ends”
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Recombinant DNA TechniqueRecombinant DNA TechniqueMutant strains of E. coli used to avoid
bacteria attacking “foreign” genesInsert insulin gene next to E. coli
B-galactosidase gene which controls transcription
Bacterial cells replicate and make copies of insulin gene
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Recombinant DNA TechniqueRecombinant DNA TechniqueInsulin protein is purified (B-galactosidase
removed)Chains are mixed and disulfide bridges
formYeast cells provide a sterile growth
mediumFinal product is Humulin - chemically
identical to human insulin
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Possible Complications of Using Possible Complications of Using Human InsulinHuman Insulinhypoglycemia (low blood sugar) tends to
be more common than with animal insulin
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The Role of Restriction The Role of Restriction EndonucleasesEndonucleasesRestriction endonucleases, first
discovered in the late 1960s, are named for preventing invasion by foreign DNA by cutting it into pieces
These enzymes cut at sites within the foreign DNA instead of chewing from the ends
By cutting DNA at specific sites they function as finely honed molecular knives
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Naming Restriction EndonucleasesNaming Restriction Endonucleases
Restriction endonucleases are named using the 1st three letters of their name from the Latin name of their source microorganism Hind III◦ First letter is from the genus H from Haemophilus◦ Next two letters are the 1st two letters of the species
name in from influenzae◦ Sometimes the strain designation is included
“d” from strain Rd
◦ If microorganism produces only 1 restriction enzyme, end the name with Roman numeral I Hind I◦ If more than one restriction enzyme is produced, the
others are numbered sequentially II, III, IV, etc.
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Restriction Endonuclease SpecificityRestriction Endonuclease Specificity
Restriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequence◦ These enzymes can recognize
4-bp, 6-bp, 8-bp sequences◦ The frequency of cuts lessens
when the recognition sequence is longer
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Restriction Enzyme TerminologyRestriction Enzyme TerminologyA 6-bp cutter will yield DNA fragments
averaging 4000-bp or 4 kilobases (4kb) in length
Heteroschizomers recognize the same DNA sequence but use a different cutting site – they are also called isochizomers
These enzymes cut DNA strands reproducibly in the same place, which is extremely useful in gene analysis
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Use of Restriction EndonucleasesUse of Restriction EndonucleasesMany restriction endonucleases make
staggered cuts in the 2 DNA strands◦ This leaves single-stranded overhangs, called
sticky ends that can base-pair together briefly◦ This makes joining 2 different DNA molecules
together much easierStaggered cuts occur when the
recognition sequence usually displays twofold symmetry, palindromes
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Restriction-Modification SystemRestriction-Modification SystemWhat prevents these enzymes
from cutting up the host DNA?◦ They are paired with methylases◦ Theses enzymes recognize,
methylate the same siteTogether they are called a
restriction-modification system, R-M system
Methylation protects DNA, after replication the parental strand is already methylated
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Basics of type II Restriction EnzymesBasics of type II Restriction Enzymes
No ATP requirement.Recognition sites in double stranded DNA have a 2-fold axis
of symmetry – a “palindrome”.Cleavage can leave staggered or "sticky" ends or can produce
"blunt” ends.
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Type II restriction enzyme Type II restriction enzyme nomenclaturenomenclature
EcoRI – Escherichia coli strain R, 1st enzymeBamHI – Bacillus amyloliquefaciens strain H, 1st enzymeDpnI – Diplococcus pneumoniae, 1st enzyme HindIII – Haemophilus influenzae, strain D, 3rd enzymeBglII – Bacillus globigii, 2nd enzymePstI – Providencia stuartii 164, 1st enzymeSau3AI – Staphylococcus aureus strain 3A, 1st enzymeKpnI – Klebsiella pneumoniae, 1st enzyme
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Why the funny names?
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An Experiment Using Restriction An Experiment Using Restriction EndonucleaseEndonuclease
An early experiment used EcoRI to cut 2 plasmids, small circular pieces of DNA independent of the host chromosome
Each plasmid had 1 site for EcoRI◦ Cutting converted circular plasmids
into linear DNA with the same sticky ends◦ The ends base pair
Some ends re-close Others join the 2 pieces
DNA ligase joins 2 pieces with covalent bonds
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Plasmids – vehicles for cloningPlasmids – vehicles for cloning
Plasmids are naturally occurring extrachromosomal DNA molecules.
Plasmids are circular, double-stranded DNA.
Plasmids are the means by which antibiotic resistance is often transferred from one bacteria to another.
Plasmids can be cleaved by restriction enzymes, leaving sticky or blunt ends.
Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid.
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Tetr
Ampr
OripBR322
4361bp
OripUC18
Ampr
MCS
LacZ
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Cloning VectorsCloning Vectors
A cloning vector is a plasmid that can be modified to carry new genes.
Plasmids useful as cloning vectors must have: An origin of replication. A selectable marker (antibiotic
resistance gene, such as ampr and tetr).
Multiple cloning site (MCS) (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers).
Easy to purify away from host DNA.
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Tetr
Ampr
OripBR322
4361bp
OripUC18
Ampr
MCS
LacZ
Older cloning vector
Newer cloning vector
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Insert – Target DNA
2. Restriction Enzymes
1. PCR product
RE1 RE2RE1
RE1
RE2RE2
T T
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VectorsVectorsVectors function as DNA carriers to allow
replication of recombinant DNAsTypical experiment uses 1 vector plus a piece
of foreign DNA ◦ Depends on the vector for its replication◦ Foreign DNA has no origin of replication, the site
where DNA replication beginsThere are 2 major classes of vectors:◦ Plasmids ◦ Phages
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Vector requirementsVector requirementsSelectable marker ◦ Resistance to ampicillin, kanamycin, hygromycin, herbicide, etc.◦ Allows only recombinant clones to survive
Multiple cloning site (mcs)◦ many (unique) restriction sites
For vectors that amplify in E. coli cells:◦ Origin of replication (Ori): required for plasmid to replicate in
bacterium◦ Control of copy number (F factor plasmid): reduces copy
number of plasmids in a given cell, lessens problems of rearrangements (chimerism)
For vectors that amplify in yeast cells:◦ Autonomous replication sequence (ARS): similar to Ori◦ CEN: centromere◦ TEL: telomere
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Plasmids As VectorsPlasmids As VectorspBR plasmids were developed early but
are rarely used todaypUC series is similar to pBR◦ 40% of the DNA, including tetracycline
resistance has been deleted◦ Cloning sites are clustered together into one
area called the multiple cloning site (MCS)
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pBR322 PlasmidpBR322 Plasmid
pBR322 illustrates cloning methods simply◦ Resistance for 2 antibiotics
Tetracycline Ampicillin
◦ Origin of replication between the 2 resistance genes◦ Only 1 site for several
restriction enzymes
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pBR322 CloningpBR322 CloningClone a foreign DNA into the PstI site of pBR322 Cut the vector to generate the sticky ends Cut foreign DNA with PstI also – compatible ends Combine vector and foreign DNA with DNA ligase to seal sticky ends Now transform the plasmid into E. coli
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Bacterial TransformationBacterial TransformationTraditional method involves incubating
bacterial cells in concentrated calcium salt solution◦ The solution makes the cell membrane leaky,
permeable to the plasmid DNANewer method uses high voltage to drive
the DNA into the cells in process called electroporation
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Transformation
Two main methods:
1. Chemical transformation – Chilling cells in the presence of Ca2+ prepares the cell walls to become permeable to plasmid DNA. Cells are briefly heat shocked which causes the DNA to enter the cell
2. Electoporation- making holes in bacterial cells, by briefly shocking them with an electric field of 10-20kV/cm. Plasmid DNA can enter the cell through these holes.
Use of bacterial cells to amplify the DNA of interest
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Screening TransformantsScreening Transformants
Transformation produces bacteria with:◦ Religated plasmid◦ Religated insert◦ Recombinants
Identify the recombinants using the antibiotic resistance◦ Grow cells with tetracycline so only cells with plasmid
grow, not foreign DNA only◦ Next, grow copies of the original colonies with ampicillin
which kills cells with plasmid including foreign DNA
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Screening With Replica PlatingScreening With Replica PlatingReplica plating transfers clone
copies from original tetracycline plate to a plate containing ampicillin
A sterile velvet transfer tool can be used to transfer copies of the original colonies
Desired colonies are those that do NOT grow on the new ampicillin plate
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pUC and pUC and ββ-galactosidase-galactosidaseNewer pUC plasmids have:◦ Ampicillin resistance gene◦ Multiple cloning site inserted into the gene lacZ’
coding for the enzyme β-galactosidase Clones with foreign DNA in the MCS disrupt the ability of
the cells to make β-galactosidase Plate on media with a β-galactosidase indicator (X-gal)
and clones with intact β-galactosidase enzyme will produce blue colonies
Colorless colonies should contain the plasmid with foreign DNA
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Lac Z gene
LacZ genepromotor
RNA pol.
Gene expression dogma
DNA
LacZ mRNARibosome
β-galactosidase
RNA
Protein
X-gal BLUE coloniesBLUE colonies
WHITE WHITE coloniescoloniesX-gal
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Expression vectors
To yield the product of a cloned gene for further studies
1) Expression vectors with a strong promoter
More mRNA More protein
2) Expression vectors with an inducible promoterForeign proteins when overexpressed could be toxicKeep the gene expression off till it is time to turn it on
a. Drug-inducible (e.g. IPTG)b. Heat-inducible
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Expression vectorsTo yield the product of a cloned gene for further studies
1) Expression vectors with a strong promoter
More mRNA More protein
2) Expression vectors with an inducible promoterForeign proteins when overexpressed could be toxicKeep the gene expression off till it is time to turn it on
a. Drug-inducible (e.g. IPTG or arabinose)b. Heat-inducible
3) Expression vectors with a fusion tag for affinity purificationFacilitate the purification of the expressed protein
1) 6 Histidine tag2) Glutathione transferase tag (GST)3) Maltose-binding protein tag
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LacZpromotor operator
Repressor
Lac Z gene
LacZpromotor
RNA pol.
RNA pol.
IPTG
IPTG
IPTG
LacZpromotor operator
IPTGIPTG
RNA pol.
IPTG
X-galΒ-galactosidase
X + galactoseCells which produce ß-galactosidase form BLUE coloniesBLUE colonies. Cells without ß-galactosidase production form WHITE WHITE coloniescolonies.
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X
XX
X
X
Plasmid without Insert
Plasmid +Insert
without plasmid
Screening
LacZ
pGEM
Insert
WHITE coloniesWHITE colonies BLUE BLUE coloniescolonies
promotor
operator
TT
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An Expression Vector for Making a LacZ Fusion Protein
LacZ can be replaced with:
1) Glutathione S-transferase (GST)
2) Maltose-binding protein (MBP)
Affinity Ligand:
Glutathione
Starch (amylose)02/20/15Asheesh Kumar Pandey ([email protected])
A plasmid DNA will be purified from the bacteria cells.
Insert
Vector
Confirmation by digestion with restriction enzyme and separation of the digestion
products on agarose gel
EcoRI
EcoRI
Plasmid DNA will be digested with EcoRI, and analyzed by gel electrophoresis for identification of the clone containing insert.
pGEM
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Directional CloningDirectional CloningCut a plasmid with 2 restriction enzymes
from the MCSClone in a piece of foreign DNA with 1
sticky end recognizing each enzymeThe insert DNA is placed into the vector
in only 1 orientationVector religation is also prevented as the
two restriction sites are incompatible
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SummarySummaryFirst generation plasmid cloning vectors include
pBR322 and the pUC plasmidspBR322 has ◦ 2 antibiotic resistance genes ◦ Variety of unique restriction sites for inserting foreign DNA◦ Most of these sites interrupt antibiotic resistance, making
screening straightforwardpUC has◦ Ampicillin resistance gene◦ MCS that interrupts a β-galactosidase gene
MCS facilitates directional cloning into 2 different restriction sites
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Relaxed, cccDNA(covalently, closed, circular)
Supercoiled DNA
Open (nicked) circular
Gyrase
Topoisomerase
Endonuclease
DNA ligase
Endonuclease
Interconversions of different forms of plasmids
Relaxed Plasmid (multiple copies per cell)
Stringent Plasmid (limited copies per cell)
Non-integrative
Episome (Integrated)
tra genes (conjugative / non-conjugative)
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Phages As VectorsPhages As VectorsBacteriophages are natural vectors that
transduce bacterial DNA from one cell to another
Phage vectors infect cells much more efficiently than plasmids transform cells
Clones are not colonies of cells using phage vectors, but rather plaques, a clearing of the bacterial lawn due to phage killing the bacteria in that area
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Vectors Host - Features Insert Size
Plasmid E.coli- Introduced by transformation 1-5 kb (electroporation or heat shock)
Phage E.coli- Virus that infects bacteria 10-15 kbIntroduced by transfection
Cosmid E.coli- Plasmid with “cos” sites for 30-45 kb packaging into lambda phage particles. Introduced by infection into E. coli
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DNA molecule originating from a virus, a plasmid, or the cell of a higher organism into which another DNA fragment of appropriate size can be integrated without loss of the vector’s capacity for self-replication
Vectors introduce foreign DNA into host cells, where the DNA can be reproduced in large quantities
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RNase H
Pol I
RNA II primes DNA synthesis
Replication
RNA II (555 bp)
RNA I (108 bp)
5’5’3’3’
Origin
Infrequently
Frequently
RNA I/ RNA II hybrid + Rop dimer (initial pairing of duplex)
Rop dimer
RNA II inactivated for primer function
No Replication
Plasmid copy number (regulation of replication of Col E1-derived plasmids
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Partitioning and segregative stability of plasmids par gene
Incompatibility of plasmidsInability of two different plasmids to coexist in the same cell in the absence of selection pressure (same mechanism of replication control)
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λλ Phage VectorsPhage VectorsFirst phage vectors were constructed by
Fred Blattner and colleagues◦ Removed middle region◦ Retained genes needed for phage replication◦ Could replace removed phage genes with
foreign DNAOriginally named Charon phageMore general term, replacement vectors
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Phage Vector AdvantagesPhage Vector AdvantagesPhage vectors can receive larger amounts
of foreign DNA◦ Charon 4 can accept up to 20kb of DNA◦ Traditional plasmid vectors take much less
Phage vectors require a minimum size foreign DNA piece (12 kb) inserted to package into a phage particle
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Cloning Using a Phage VectorCloning Using a Phage Vector
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Genomic LibrariesGenomic LibrariesA genomic library contains clones of all
the genes from a species genomeRestriction fragments of a genome can be
packaged into phage using about 16 – 20 kb per fragment
This fragment size will include the entirety of most eukaryotic genes
Once a library is established, it can be used to search for any gene of interest
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Plaque HybridizationPlaque Hybridization
Searching a genomic library requires probe showing which clone contains desired gene
Ideal probe – labeled nucleic acid with sequence matching the gene of interest
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CosmidsCosmidsCosmids are designed for cloning large DNA fragments◦ Behave as plasmid and phage◦ Contain
cos sites, cohesive ends of phage DNA that allow the DNA to be packaged into a λ phage head
Plasmid origin of replication permitting replication as plasmid in bacteria
◦ Nearly all λ genome removed so there is room for large inserts (40-50 kb)◦ So little phage DNA can’t replicate, but they are infectious
carrying recombinant DNA into bacterial cells
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M13 Phage VectorsM13 Phage VectorsLong, thin, filamentous phage M13Contains:◦ Gene fragment with β-galactosidase◦ Multiple cloning site like the pUC family
Advantage◦ This phage’s genome is single-stranded DNA◦ Fragments cloned into it will be recovered in
single-stranded form
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M13 Cloning to Recover Single-M13 Cloning to Recover Single-stranded DNA Productstranded DNA Product
After infecting E. coli cells, single-stranded phage DNA is converted to double-stranded replicative form
Use the replicative form for cloning foreign DNA into MCS
Recombinant DNA infects host cells resulting in single-stranded recombinant DNA
Phage particles, containing single-stranded phage DNA is secreted from transformed cells and can be collected from media
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PhagemidsPhagemids
Phagemids are also vectors◦ Like cosmids have aspects of
both phages and plasmids◦ Has a MCS inserted into lacZ’
gene to screen blue staining / white colonies◦ Has origin of replication of
single-stranded phage f1 to permit recovery of single-stranded recombinant DNA◦ MCS has 2 phage RNA
polymerase promoters, 1 on each side of MCS
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Bacterial Artificial Chromosomes(BACs) and Bacterial Artificial Chromosomes(BACs) and Yeast Artificial Chromosomes(YACs)Yeast Artificial Chromosomes(YACs)
BACs can hold up to 300 kbs. The F factor of E.coli is capable of
handling large segments of DNA. Recombinant BACs are introduced
into E.coli by electroportation ( a brief high-voltage current). Once in the cell, the rBAC replicates like an F factor.
Example: pBAC108L Has a set of regulatory genes, OriS,
and repE which control F-factor replication, and parA and parB which limit the number of copies to one or two.
A chloramphenicol resistance gene, and a cloning segment.
YACs can hold up to 500 kbs. YACs are designed to replicate as
plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted, YACs are transferred to cells, they then replicate as eukaryotic chromosomes.
YACs contain: a yeast centromere, two yeast telomeres, a bacterial origin of replication, and bacterial selectable markers.
YAC plasmidYeast chromosome DNA is inserted to a unique restriction
site, and cleaves the plasmid with another restriction endonuclease that removes a fragment of DNA and causes the YAC to become linear. Once in the cell, the rYAC replicates as a chromosome, also replicating the foreign DNA.
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PROPERTY P1 pBAC pucBAC pCYPAC YAC
Vector Size (kb) 31 6.5 7.2 19.3 11.5
Vector Copy # single single multiple multiple multiple
Insert Size (kb) 75-95 0-300 0-300 0-300 0-2000
Cloning Strategy 2 arms single digest single digest single digest double digest
BamHI/ScaI Bam or Hind Bam or Hind Bam/Sca link Bam/EcoRI
Cloning Method Packaging Electroporate Electroporate Electroporate Spheroplast
Maintenance (copy #) single single single single single
Chimeric Clones (%) 0 2 2 0 (24/24) 20-60
Positive Selection yes no no yes yes
Copy # Induction yes no no yes no
Large Fragment Cloning VectorsLarge Fragment Cloning Vectors
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Selected Protein Expression SystemsLiving Hosts:
Escherichia coli
Saccharomyces cerevisiae
Picchia pastoris
Baculovirus
Mammalian tissue culture
Transgenic animals (flies, mice, barnyard animals)
Specialized Systems:
Xenopus oocytes
In vitro transcription-translation
Choosing a System:
-- yield
-- product purification
-- post-translational modifications
-- genetic manipulation of gene/cDNA
-- cost
-- experimental difficulty02/20/15
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SummarySummaryTwo kinds of phage are popular cloning vectors
‑ λ phage ‑ Has nonessential genes removed making room for inserts- Cosmids accept DNA up to 50 kb
- M13 phage- Has MCS- Produces single-stranded recombinant DNA
Plasmids called phagemids also produce single-stranded DNA in presence of helper phage
Engineered phage can accommodate inserts up to 20 kb, useful for building genomic libraries
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Eukaryotic Vectors and Very Eukaryotic Vectors and Very High Capacity VectorsHigh Capacity Vectors
There are vectors designed for cloning genes into eukaryotic cells
Other vectors are based on the Ti plasmid to carry genes into plant cells
Yeast artificial chromosomes (YAC) and bacterial artificial chromosomes (BAC) are used for cloning huge pieces of DNA
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Identifying a Specific Clone With Identifying a Specific Clone With a Specific Probea Specific Probe
Probes are used to identify a desired clone from among the thousands of irrelevant ones
Two types are widely used◦ Polynucleotides also called oligonucleotides ◦ Antibodies
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Polynucleotide ProbesPolynucleotide Probes
Looking for a gene you want, might use homologous gene from another organism◦ If already cloned◦ Hope enough sequence similarity to permit
hybridization◦ Need to lower stringency of hybridization
conditions to tolerate some mismatches
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Control of Hybridization Control of Hybridization StringencyStringency
Factors that promote separation of two strands in a DNA double helix:◦ High temperature◦ High organic solvent concentration◦ Low salt concentration
Adjust conditions until only perfectly matched DNA strands form a duplex = high stringency
Lowering these conditions lowers stringency until DNA strands with a few mismatches can hybridize
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SummarySummarySpecific clones can be identified using
polynucleotide probes binding to the gene itself
Knowing the amino acid sequence of the a gene product permits design of a set of oligonucleotides that encode part of the amino acid sequence
Can be a very quick and accurate means of identifying a particular clone
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cDNA CloningcDNA CloningcDNA is the abbreviation for
complementary DNA or copy DNAA cDNA library is a set of clones
representing as many as possible of the mRNAs in a given cell type at a given time◦ Such a library can contain tens of thousands
of different clones
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Reverse Transcriptase PrimerReverse Transcriptase PrimerCentral to successful cloning is the
synthesis of cDNA from an mRNA template using reverse transcriptase (RT), RNA-dependent DNA polymerase◦ RT cannot initiate DNA synthesis without a
primer◦ Use the poly(A) tail at 3’ end of most
eukaryotic mRNA so that oligo(dT) may serve as primer
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Ribonuclease HRibonuclease HRT with oligo(dT) primer has made a
single-stranded DNA from mRNANeed to start to remove the mRNAPartially degrade the mRNA using
ribonuclease H (RNase H)◦ Enzyme degrades RNA strand of an RNA-DNA
hybrid◦ Remaining RNA fragments serve as primers
for “second strand” DNA using nick translation
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Nick TranslationNick TranslationThe nick translation process
simultaneously:◦ Removes DNA ahead of a nick◦ Synthesizes DNA behind nick◦ Net result moves or translates the
nick in the 5’ to 3’ directionEnzyme often used is E. coli
DNA polymerase I◦ Has a 5’ to 3’ exonuclease activity ◦ Allows enzyme to degrade DNA
ahead of the nick
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Trailing Terminal TransferaseTrailing Terminal Transferase
Don’t have the sticky ends of genomic DNA cleaved with restriction enzymes
Blunt ends will ligate, but inefficientGenerate sticky ends using terminal
deoxynucleotidyl transferase (TdT), terminal transferase with one dNTP◦ If use dCTP with the enzyme◦ dCMPs are added one at a time to 3’ ends of the cDNA◦ Same technique adds oligo(dG) ends to vector◦ Generate ligation product ready for transformation
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Vector ChoiceVector ChoiceChoice based on method used to detect
positive clonesPlasmid or phagemid like pUC or pBS will
be used with colony hybridization and a labeled DNA probe
If λ phage like λgt11, cloned cDNA under control of lac promoter for transcription and translation of the cloned gene and antibody screening
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Rapid Amplification of cDNA Rapid Amplification of cDNA EndsEndsIf generated cDNA is not full-length,
missing pieces can be filled in using rapid amplification of cDNA ends (RACE)
Technique can be used to fill in either the missing portion at the 5’-end (usual problem)
Analogous technique can be used to fill in a missing 3’-end
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5’-RACE5’-RACEUse RNA prep containing
mRNA of interest and the partial cDNA
Anneal mRNA with the incomplete cDNA
Reverse transcriptase will copy rest of the mRNA
Tail the completed cDNA with terminal transferase using oligo(dC)
Second strand synthesis primed with oligo(dG)
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SummarySummaryMake cDNA library with synthesis of cDNAs one strand at
a time◦ Use mRNAs from a cell as templates for 1st strands, then 1st
strand as template for 2nd
◦ Reverse transcriptase generates 1st strand◦ DNA polymerase I generates the second strands
Give cDNAs oligonucleotide tails that base-pair with complementary tails on a cloning vector
Use these recombinant DNAs to transform bacteriaDetect clones with:◦ Colony hybridization using labeled probes◦ Antibodies if gene product translated
Incomplete cDNA can be filled in with 5’- or 3’-RACE
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Methods of Expressing Cloned Methods of Expressing Cloned GenesGenesCloning a gene permits Production of large quantities of a
particular DNA sequence for detailed study
Large quantities of the gene’s product can also be obtained for further use◦ Study ◦ Commerce
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Expression VectorsExpression Vectors
Vectors discussed so far are used to first put a foreign DNA into a bacterium to replicate and screen
Expression vectors are those that can yield protein products of the cloned genes◦ For high level expression of a cloned gene best
results often with specialized expression vectors◦ Bacterial vectors have a strong promoter and a
ribosome binding site near ATG codon
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Fusion ProteinsFusion Proteins
Some cloning vectors, pUC and pBS, can work as expression vectors using lac promoter
If inserted DNA is in the same reading frame as interrupted gene, a fusion protein results◦ These have a partial β-
galactosidase sequence at amino end◦ Inserted cDNA protein
sequence at carboxyl end02/20/15
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Inducible Expression VectorsInducible Expression VectorsMain function of expression vector is to yield the
product of a gene – usually more is betterFor this reason, expression vectors have very strong
promotersPrefer keep a cloned gene repressed until time to
express◦ Large quantities of eukaryotic protein in bacteria are
usually toxic◦ Can accumulate to levels that interfere with bacterial
growth◦ Expressed protein may form insoluble aggregates, inclusion
bodies
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Controlling theControlling the lac lac Promoter Promoterlac promoter is somewhat inducible◦ Stays off until stimulated◦ Actually repression is incomplete or leaky◦ Some expression will still occur
To avoid this problem, express using a plasmid or phagemid carrying its own lacI repressor gene, such as pBS
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Arabinose PromoterArabinose PromoterThe hybrid trc promoter combines
strength of the trp (tryptophan operon) promoter with inducibility of lac promoter
Promoter from ara operon, PBAD, allow fine control of transcription◦ Inducible by arabinose, a sugar◦ Transcription rate varies with arabinose
concentration
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Tightly Controlled PromoterTightly Controlled Promoter
Lambda (λ) phage promoter, PL, is tightly controlled
Expression vectors with this promoter-operator system are used in host cells with temperature-sensitive λ repressor gene◦ Repressor functions are low temperatures◦ Raise temperature to nonpermissive
temperature, the repressor doesn’t function and cloned gene is expressed
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02/20/15Asheesh Kumar Pandey ([email protected])
cI repressor
Bacterial chromosome
Bacterial chromosome
Po Ptrp λcI repressor
Transcription
No Transcription
Po PL ATG Gene of Interest
Vector
Vector
Tryptophan
trp repressor
No Transcription
λcI repressor
Transcription
ATG Gene of Interest
Po
Po
Ptrp
PL
+ Tryptophan
- TryptophanRegulation of gene expression
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SummarySummaryExpression vectors are designed to yield
the protein product of a cloned geneWhen a lac inducer is added, cell begins
to make T7 polymerase which transcribes the gene of interest
Many molecules of T7 polymerase are made, so gene is turned on to a very high level with abundant amount of protein product made
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Expression Vectors That Produce Expression Vectors That Produce Fusion ProteinsFusion Proteins
Most vectors express fusion proteins◦ The actual natural product of the gene isn’t made◦ Extra amino acids help in purifying the protein product
Oligohistidine expression vector has a short sequence just upstream of MCS encoding 6 His◦ Oligohistidine has a high affinity for divalent metal ions like
Ni2+ ◦ Permits purification by nickel affinity chromatography◦ His tag can be removed using enzyme enterokinase without
damage to the protein product
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Oligohistidine Expression VectorOligohistidine Expression Vector
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Fusion Proteins in Fusion Proteins in λλgt11gt11
This phage contains lac control region and lacZ gene
Products of gene correctly inserted will be fusion proteins with a β-galactosidase leader
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Antibody Screening With Antibody Screening With λλgt11gt11Lambda phages with cDNA
inserts are platedProtein released are
blotted onto a supportProbe with antibody to
proteinAntibody bound to protein
from plaque is detected with labeled protein A
Partial cDNAs can be completed with RACE
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SummarySummaryExpression vectors frequently produce fusion
proteins◦ One part of the protein comes from coding
sequences in the vector◦ Other part from sequences in the cloned gene
Many fusion proteins have advantage of being simple to isolate by affinity chromatography
Vector lgt11 produces fusion proteins that can be detected in plaques with a specific antiserum
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Bacterial Expression System Bacterial Expression System ShortcomingsShortcomingsThere are problems with expression of
eukaryotic proteins in a bacterial system◦ Bacteria may recognize the proteins as foreign and
destroy them◦ Posttranslational modifications are different in
bacteria◦ Bacterial environment may not permit correct
protein foldingVery high levels of cloned eukaryotic proteins
can be expressed in useless, insoluble form
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Eukaryotic Expression SystemsEukaryotic Expression SystemsAvoid bacterial expression problems by
expressing the protein in eukaryotic cellInitial cloning done in E. coli using a shuttle
vector, able to replicate in both bacterial and eukaryotic cells
Yeast is suited for this purpose◦ Rapid growth and ease of culture◦ Still a eukaryote with more appropriate
posttranslational modification◦ Secretes protein in growth medium so easy
purification02/20/15
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Use of Baculovirus As Expression Use of Baculovirus As Expression VectorVector
Viruses in this class have a large circular DNA genome, 130 kb
Major viral structural protein is made in huge amounts in infected cells◦ Promoter for this protein, polyhedrin, is very active◦ These vectors can produce up to 0.5 g of protein
per liter of medium◦ Nonrecombinant viral DNA entering cells cannot
result in infectious virus as it lacks an essential gene supplied by the vector
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Animal Cell TransfectionAnimal Cell TransfectionCalcium phosphate◦ Mix cells with DNA in a phosphate buffer◦ Then solution of calcium salt added to form a
precipitate◦ Cells take up the calcium phosphate crystals which
include some DNALiposomes◦ DNA mixed with lipid to form liposomes, small vesicles
with some of the DNA inside◦ DNA-bearing liposomes fuse with cell membrane
carrying DNA inside the cell
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Choosing a Mammalian Cell Expression SystemAdvantages:
Cell biology (ie. Localization, cell cycle etc.)
Post-translational modification (mammalian)
Improving expression vectors and transfection methods
Disadvantages:
Low yield of expressed proteins
Relatively expensive (tissue culture costs)
Must use shuttle vectors
Key terms:
Transient transfection: introduction of episomal expression vector into mammalian tissue culture cells for short term expression experiments.
Stable transfection: introduction and integration of vector into the host cell chromosome for long-term expression studies.
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02/20/15Asheesh Kumar Pandey ([email protected])
02/20/15Asheesh Kumar Pandey ([email protected])
02/20/15Asheesh Kumar Pandey ([email protected])
SummarySummary
Foreign genes can be expressed in eukaryotic cells
These eukaryotic systems have advantages over prokaryotic ones◦ Made in eukaryotic cells tend to fold properly and
are then soluble rather than aggregated into insoluble inclusion bodies◦ Posttranslational modifications are made in a
eukaryotic manner
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Using the Ti Plasmid to Transfer Using the Ti Plasmid to Transfer Genes to PlantsGenes to Plants
Genes can be introduced into plants with vectors that can replicate in plant cells
Common bacterial vector promoters and replication origins are not recognized by plant cells
Plasmids are used containing T-DNA◦ T-DNA is derived from a plasmid known as tumor-
inducing (Ti)◦ Ti plasmid comes from bacteria that cause plant
tumors called crown galls
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Ti Plasmid InfectionTi Plasmid InfectionBacterium infects plant, transfers Ti
plasmid to host cellsT-DNA integrates into the plant DNA
causing abnormal proliferation of plant cells
T-DNA genes direct the synthesis of unusual organic acids, opines which can serve as an energy source to the infecting bacteria but are useless to the plant
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Ti Plasmid Transfers Crown GallTi Plasmid Transfers Crown Gall
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