Basis of course
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Basis of course
• Understand the technology• Understand the terminology• Gain some practical experience• The applications in biotechnology and basic biology next
year• Why?
– Fluidity next year (don’t need to explain terms and technology while discussing the applications)
– Shows what molecular biology projects are like• Disadvantage
– Technology dominated. Can make it a bit boring
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Lectures
• Two per week, however– The Tuesday lecture may be used to
discuss data from practicals– In some lecture slots the in course test will
be held– Approximately formal 15 lectures
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Practicals
• I will supervise the first two, Dianne Ford the second two
• Objectives of practicals– Get used to pipetting small quantities– Reinforce lectures– Help focus on project choice for nexy year– Schedules soon
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BNS216• Phases of course
– Isolation of a specific DNA sequence (gene or cDNA)
– Analysis of isolated DNA sequence • DNA sequencing
– Manipulation of DNA sequence• PCR to introduce restriction enzyme site• PCR to change codon• PCR to detect specific DNA sequence
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BNS216– Production of recombinant protein
• Expression vectors
– Detecting genes and transcripts• Northern hybridization• Southern hybridization• RTPCR
– Manipulation of eukaryotic organisms
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BNS216
• Isolation of a gene– Construction of a gene library
• Choice or organism• Restriction enzymes• DNA ligase• Vectors and which ones to use• Screening startegies
– Prokaryotic and you look for the protein product– Eukaryotic and you look for the DNA
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BNS216
• Isolation of a cDNA– Purification of mRNA– DNA from mRNA– Construction cDNA library– Screening the library Analysis of DNA
• DNA sequencing• Why you need to know the sequence• Automated method with fluorescent
dideoxynucleotides
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BNS216
• Manipulation of DNA– PCR and how it relies on knowing the sequence
• Introduction of restriction enzyme sites• Changing the amino acid sequence of a protein • Expression of proteins in bacteria
• Expression vectors– How they enable foreign genes or cDNAs to be
expressed– How expression vectors are regulated
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BNS216• Detecting transcription and genes
– Northern hybridization– RTPCR– Southern hybridization
• Manipulation of eukaryotic multicellular organisms– Transgenic animals
• Insertion of foreign genes by microinjection• Inactivating genes by homologous recombination in stem
cels
– Transgenic plants
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Assessment
• Exam:– 60 % of module– Answer 3 questions from 5– Must get >35 % to pass module
• Practicals– 20 % of module– Four practicals starting on 4th February
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Assessment
• In course tests– Four tests
• Test one: Isolation of a gene or cDNA• Test two: DNA sequencing and PCR• Test three: Expression vectors and nucleic acid
detection • Test four: Manipulating eukaryotic organisms
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BNS216 references• Difficult!
– Gene Cloning: An introduction T.A. Brown OK quite simple but not in same way I teach it
– Principles of Gene Manipulation: An introduction to genetic engineering R.W. Old and S.B. Primrose. Quite detailed, some of which is unnecessary
– Use any standard molecular biology or genetics text book, there will a section on BNS216
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What is genetic engineering or recombinant DNA technology?
• A suite of technologies that enable you to • Isolate and characterise genes• Produce and characterise proteins• Alter the genetic make up of an organism
– New genes– Loss of existing genes
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Applications of genetic engineering
• Basic understanding of biology– Protein structure and function– Regulation of gene expression– Importance of proteins in whole organisms
(gene knockouts or null mutations)
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Applications of genetic engineering
• Practical applications– Production of industrially important proteins– Change the properties of proteins– Modification of the phenotype of whole
organisms– Diagnosis– Primary applications in medicine and agriculture– Others include chemical, paper and detergent
industries
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Applications
• More details on applications
• Protein production
• Pharmaceutical proteins.– Constant supply and safe– Growth hormone, insulin, Factor VIII and
IX, antitrypsin
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Proteins
• Microbial proteins• Microbes grow poorly but produce
valuable enzymes– Hyperthermophiles– Anaerobes– Archaebacteria
• Genetic engineering makes these proteins available to industry
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Enzymes used in the Food Industry
• Glucose isomerase (Food industry)• Xylanases (Paper industry) • Cellulases (Energy and detergent
industries) • Phytases (animal feed)• Protein engineering
– Rational design– Forced protein evolution
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Modifying organisms
• Genetically engineered foods– Herbicide resistance– Pesticide resistance
• Good for consumer, farmer or biotech. Companies?– Golden rice with increased vitamin A and oil
seed rape with better polyunsaturated fats
• Good for consumer?
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Genetically engineered foods
• Risks
– The environment, spread of resistant weeds, alter ecological balance?
– Human health. Will we get increased antibiotic resistance
– Will the transgene be deleterious to human health?
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Change phenotype of farm animals
• Convert them into bioreactors to produce pharmaceutical proteins. Why?
• Change their biochemistry so• More efficient use of nutrients• Better quality end-products e.g. milk and
meat– Humanising milk– Increase polyunsaturates
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Change phenotype of small animals
• Generate animals for human disease influenced by diet
• Colon cancer
• nvCJD
• Heart disease
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Gene therapy
• Correct genetic defect– Not in germ line– Not transmissible
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Diagnosis
• Diagnosis
– Human genome sequenced
– Identify all genes soon
– Immediate diagnosis test
• Hungtintons
• Muscular dystrophy
• Cystic fibrosis
• Sickle cell anaemia
• Alzeihmer
• Breast cancer
• Colon cancer
• Heart disease
– Good or bad?
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Diagnosis• Reduce incidence of disease
– Pregnancy termination– Pre-implantation selection
• Start treatment to prevent disease– Prophylactic mastectomy– Colon removal– Physiotherapy
• Stress if diagnosed. Do you want to know?
• Insurance and job prospects?
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Genetic engineering history
• Pioneered by Cohen and Boyer 1972-1974 (bacterial systems)
• Southern hybridization 1975
• DNA sequencing 1977-1980
• Transgenic animals 1980
• Polymerase chain reaction 1985
• Site-directed mutagenesis 1985
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Where do we start?
• If we want to do genetic engineering how do we start?
• Isolate the gene of interest– Select organism containing gene– Construct a gene library– Select members of the gene library that
contain the gene of interest
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How do you start doing recombinant DNA technology?
• Isolate the gene of interest
• Lets isolate (clone) a cellulase gene– Identify organism that contains the gene
• Rumen• compost• Soil• Leaf litter• Decaying wood
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Isolating a cellulase gene
Isolate chromosomalDNA
Fragment DNA
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Gene library
E. coli
Vector
Mix and ligate
Transform
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Properties of vector DNA• Replicates in bacterium
• Foreign DNA inserted will be stable
• Normally extra-chromosomal
• Easy to select bacterium containing vector (confers antibiotic resistance)
• Vectors– Plasmid (extra chromosomal circular DNA)– Bacteriophage– Cosmids– Artificial chromosomes
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Gene library
Screen libraryfor appropriate gene(cellulase gene)
Isolate plasmid
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Isolating a cellulase gene
Isolate chromosomalDNA
Fragment DNA
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Restriction endonucleases• Enzymes that cut DNA at specific
sequences
• Discovered in the early 1950s
• Agent that enables bacteria to be immune to bacteriophage
• Host-controlled restriction
• Mainly found in bacteria
• Over 1200 characterised
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Restriction endonucleases
• Three types only Type II important in genetic engineering as they cut the sequence they recognise
• Target sequences generally palindromic
• Recognise 4, 6 or 8 nucleotides
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Restriction endonucleases
GAATTCCTTAAG
CTGCAGGACGTC
CCCGGGGGGCCC
G AATTCCTTAA G
CTGCA GG ACGTC
CCC GGGGGG CCC
EcoRI
PstI
SmaI
Sticky ends
Blunt ends
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Restriction endonucleases
• Named after organism• e.g. EcoRI = Escherichia (E) coli (co) strain R (R). I
refers to Ist enzyme isolated from organism• Why doesn’t a bacterial restriction endonuclease
digest its own DNA?• The bacterium produces a DNA methylase that
recognises same sequence as restriction endonuclease
• Methylates target DNA sequence which makes it resistant to endonuclease cleavage
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Restriction enzymes and DNA methylase
GAATTCCTTAAG
GAATTCCTTAAG
GAATTCCTTAAG
CH3
CH3
GAATTCCTTAAG
CH3
CH3
EcoRI DNA methylase
EcoRI
G AATTCCTTAA G
Foreign DNA Host DNA
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Gene library
E. coli
Vector
Mix and ligate
Transform
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GAATTCCTTAAG
GAATTCCTTAAG
G AATTCCTTAA G
G AATTCCTTAA G
GAATTCCTTAAG
GAATTCCTTAAG
GAATTCCTTAAG
Forming hybrid or recombinant DNA moleculesusing restriction enzymes and DNA ligase
GAATTCCTTAAG
GAATTCCTTAAG
GAATTCCTTAAG
Recombinant or hybrid DNA
DNA ligaseDNA ligase
Digest with EcoRI
Mix DNA
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Inserting chromosomal DNA into a vector
GAATTCCTTAAG
GAATTCCTTAAG
GAATTCCTTAAG
GAATTCCTTAAG
GAATTCCTTAAG
Vector
Chromosome
Cut with EcoRI and add DNA ligase
Recombinant vector
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More details on each stage
• Chromosomal DNA is only partially cut because?
• Don’t know if the restriction enzyme cuts in the gene
• Plasmid vector is designed to enable selection for recombinant plasmid– pUC or pBluescript-based plasmid vectors– Contains two selection genes ampicillin
(antibiotic) and LacZ; codes for -galactosidase
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pUC18
LacZ’ encodes -galactosidase
Ampr confers ampicillin resistance
Origin of replication
EcoRIHindIII BamHI
Cells containingpBluescript areampicillin resistanceand blue on X-Gal
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Bromo-chloro-indoyl--galactopyranosidase or X-Gal (Clear)
Bromo-chloro-indoyl (Deep blue insoluble)
+
galactose
-galactosidase
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Inserting chromosomal DNA into a vector
GAATTCCTTAAG
GAATTCCTTAAG
GAATTCCTTAAG
GAATTCCTTAAG
GAATTCCTTAAG
Vector
Chromosome
Cut with EcoRI and add DNA ligase
Recombinant vector
Ampicillin resistant; -galactosidase negative (White on X-Gal)
LacZ gene codes for -galactosidase
Ampicillin resistance gene
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GAATTCCTTAAG
Wild type vector
Ampicillin resistant; -galactosidase active (Blue on X-Gal)
LacZ gene codes for -galactosidase
Ampicillin resistance gene
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E. coli sensitive to ampicillin
Ampicillin resistant; -galactosidase active (Blue on X-Gal)
Ampicillin resistant; -galactosidase negative (White on X-Gal)
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Bacteria from ligation platedon ampicillin and X-Gal
Containswild typeplasmd
Containsrecombinantplasmd
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Gene library
• Collection of microbes (e.g. Escherichia coli) each one containing a recombinant vector
• Each recombinant vector contains a random region of the target chromosome
• The number of microbes in the library is large• Thus any gene in the target organism’s
genome is present in at least one member of the gene library
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Gene library
E. coli
Vector
Mix and ligate
Transform
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Size of gene library
N = ln(1-P) ln (1-A/B)
N = Number of clones P = 95 % probability of finding geneA = Average size of DNA fragmentsB = Total size of genome
E. coli has genome of 4,800,000 nucleotidesAverage size of insert is 10,000 nucleotidesNumber of clones for 95 % probability is 1700
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Size of gene library
• If genome is large e.g. human genome (3 x 109) then number of clones to make library becomes unrealistic (1058000) if using a plasmid vector (accepts only 10 kb as larger DNA can’t be transformed)
• Therefore need to use vectors that can accept larger pieces of DNA– I.e. if each vector contains a large piece of DNA you
don’t need so many clones to make a gene library
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Vectors that accept larger DNA
• Plasmid: 10 kb
• Lambda bacteriophage: 18-25 kb
• Cosmid: ~40 kb
• Yeast or bacterial artificial chromosome: 100-1000 kb
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Gene library
Screen libraryfor appropriate gene(cellulase gene)
Isolate plasmid
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Screening gene library for cellulase gene
• Assume bacterial genes will express in Escherichia coli
• Escherichia coli does not degrade polysaccharides
• Screen library by looking for members that degrade cellulose
• Similar approach for other polysaccharidases (amylases, pectinases, xylanases etc)
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Vectors
• Lambda vector• Infects E. coli replicates and then viruses
released• End of genome are 12 bp sequences
known as cos sequences. • Cos sequences play an important role in
packaging viral DNA into capsids (head of the virus)
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DNA replication generatingconcatamers
Lambda infects E. coli
DNA injectedinto E. coli
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Lambda DNA is linear in virusCos sequence is 12 nucleotides and single strandedThe two cos sequences are complementary
Replicates by rolling ciricle in E. colito produce concatemerscos lambda DNA cos etc etc
cos sequences hybridise in E.colito form circular genome
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Lambda head genes transcribed and translatedto produce head proteins
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Endoglucanase A cuts at cossequence
Endoglucanase A expressed. Cuts DNA at cos sequenceand assists packaging lambda DNA into viral capsid(head proteins)
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Tail genes then expressed
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Tail bind to heads to form virus
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Lambda virus produces lysozyme that hydrolysesbacterial cell wall releasing viruses to attack other bacterial cells
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DNA replication generatingconcatamers
Lambda infects E. coli
DNA injectedinto E. coli
Replication by a mechanism called rolling circle
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Vectors
• More information on vectors• Lambda vector• Libraries contain larger inserts than
plasmids (20-25 kb)• Naked Lambda DNA can’t be transformed
into E. coli• Lambda DNA can be packaged into a virus• Virus then infected into E. coli
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In vitro packagingTwo E. coli mutants. One synthesises the tails the other the heads and A protein1. Take DNA and mix with E. coli extracts containing heads and A protein
2. Packaged DNA is then mixed with E. coli extracts containing tails
3. The virulent phage can then be used to infect E.coli to form plaques in a lawn of bacteria
+
Cos sequence
Internal DNA
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Lambda vector for genomic cloning
1.Lambda genome is 40 kb
2. Lambda vectors contain right arm, left arm and central region. At ends are single stranded cos sequences
3. Genes in central region not essential
4. Restriction sites at boundary of central region and the two arms.
5. Used to clone DNA about 20 kb
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Cloning DNA into vectors
Left arm Central Right arm Chromosomal DNA
Cut out 3 regionsand purify arms
Cut chromosome withsame enzyme as lambda
Mix DNA and addDNA ligase
Will not package Recombinant lambda will package. Why?
cos
DNA will formconcatamers
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DNA packaging is size dependent
DNA too large to package >25 kb
DNA packages 18-25 kb
DNA too smallto package <18 kb
cos
Endonuclease Acleaves cos sequence
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Cosmid vectors• Problem with vectors
is that you can’t transform large pieces of DNA into E. coli
• Cosmid– Similar to plasmid
• Ampicillin resistance gene
• 5 kb in size• Unique BamHI site• Cos sequence
Cos
Ampr
BamHI
Origin
Cosmid vector
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Cosmid vectors
• Accepts as much as 40 kb of chromosomal DNA
• Why?– Smuggle cosmid into E. coli by packaging it
into lambda virus as it has a cos sequence
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Cos
Ampr
BamHI
GGATCC GGATCCCCTAGG CCTAGG
GATCC G G CCTAG
GGATCC GGATCC GGATCC GGATCC
GGATCC GGATCC GGTACC GGATCC
Cut with BamHI
GCCTAG
GATCC G
Cut with BamHI
Mix DNA at veryhigh concentrationand add DNA ligase
Origin
Packaged in vitro
Cosmid vector
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How do you selectfor E. coli cells containingcosmids?
They are resistantto ampicillin
Lambda particleinjects cosmid in E. coli.E. coli is viable asno lambda genes in cosmid so it acts asa normal plasmid
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Origins of
replication
CentromereTelomere Telomere
Accepts up to 1 Mb of chromosomal DNA
BamHI sites
sup4 gene
centromereoriginof replication
Trp1gene
SnaBI site
Yeast Artificial Chromosome (YAC)Accepts up to 1 Mb of DNA
Telomeres
Ura3 gene
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0.1-1 Mb
Telomere TelomereTrp1 Ori Cent sup4’ chromosomal DNA sup4’ Ura3
Cut withSnaBI andBamHI
Mix and addDNA ligase
Transform recombinant YACs into mutant yeastthat lack Ura3 and Trp1 genes (I.e. can’t make tryptophanand uracil) so the amino acid and nucleotide mustbe added for yeast to grow
Cut with SnaBI
Telomeres
BamHI sites
sup4 gene
Ura3 gene
centromereoriginof replication
Trp1gene
SnaBI site
Yeast Artificial Chromosome Chromosomal DNA
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Plate yeast on medium lacking uracil and tryptophan. Yeast colonies that grow contain YACand those that are pink contain recombinant YACas this indicates inactivation of Sup 4
Colony containswild type YAC
Colony containsrecombinant YAC
Transformed yeast plated on media lacking tryptophan and uracil
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What vectors for what libraries?
Vector Insertsize
What libraries
Plasmid <10 kb Bacteria
Lambda phage 18-25 kb Yeast
Cosmid 34-45 kb Intermediateeukaryotes
YAC etc 0.1 – 1 Mb Higher Eukaryotes
Human library requires 14000 YAC clones
Human library requires >1,000,000 plasmid clones
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Screening bacterial gene library for a specific gene
• Assume bacterial genes will express in Escherichia coli
• Screen for gene by looking for protein that– Changes phenotype of E. coli
• e.g. confers ability to degrade a polysaccharide (cellulase, xylanase etc) confers green fluorescence (green fluorescent protein)
– Changes phenotype of an E. coli mutant. Known as complementation
– Screen for protein directly using an antibody
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Complementation• Isolate gene from a bacterium that E. coli
contains– e.g. Isolate B. subtilis gene coding 1st enzyme in
leucine biosynthsis pathway
• Assume bacteria have same pathway for leucine synthesis
• Step 1: Isolate a mutant of E. coli unable to synthesise leucine – I.e. gene for 1st enzyme non-functional thus enzyme
not produced
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Add mutagen
E. coli culture Mutated E.coli culture
Plate out on media containing
leucine
Transfercoloniesto medialacking leucine
Colony 4 requiresleucine for growth(leucine auxotroph)
Complementation(generating a leucine auxotroph)
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Complementation
• Leucine auxotrophs isolated require leucine for growth– Defect in gene coding for leucine synthesis
• Which gene in pathway mutated?
• Assay leucine auxotrophs for mutant lacking enzyme one
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Complementation
• Construct a gene library of Bacillus subtilis chromosomal DNA– Digest chromosome– Insert into E. coli vector to make a library of
recombinant vectors– Transform these recombinant vectors into
the E. coli leucine auxotroph lacking enzyme 1
– Plate out library on media lacking leucine
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Complementation
• E. coli colony that grows contains recombinant plasmid with Bacillus leucine biosynthetic gene that codes for 1st enzyme in leucine synthesis
• The recombinant plasmid has overcome or complemented the E. coli mutation
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Antibody screening
• Some bacterial genes code for proteins that don’t change phenotype of E. coli wild type or mutant e.g. asparaginase from Erwinia
• Erwinia asparaginase converts asparagine to aspartic acid
• Used as a major ingredient of chemotherapy of childhood acute leukaemia
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Antibody screening
• Porton needed to produce more to supply world demand– Overexpress enzyme in E. coli
• How do you isolate gene?• Make a gene library of Erwinia DNA in E. coli• Screen library using antibody specific to
Erwinia asparaginase• How do you produce antibody?
![Page 86: Basis of course](https://reader036.fdocuments.in/reader036/viewer/2022062407/56812e5d550346895d9403ce/html5/thumbnails/86.jpg)
Purify asparaginasefrom Erwinia
Blood of rabbit contains antibodies to Erwinia asparaginase butno other Erwinia protein
Erwinia
Antibody Production
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1 2 3
4 5 6
7 8 9
Screening E. coli library of Erwinia DNA
Plate out E. colicells on nylon filter
1 2 3
4 5 6
7 8 9
Lyse bacteria with lysozymeProtein binds to filter and thus a print of the bacterium’sproteins replaces colony
1 2 3
4 5 6
7 8 9
Add inert protein(e.g. bovine serumalbumin) to blockprotein binding siteson nylon filter
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1 2 3
4 5 6
7 8 9
Add antibodieslabelled withradioactive iodine
1 2 3
4 5 6
7 8 9
1 2 3
4 5 6
7 8 9
Expose filter to X-ray filmto detect where antibody has bound (radioactive andthus blackens X-ray film)
Screening E. coli library of Erwinia DNA
www.staff.ac.ncl/h.j.gilbert
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Practical information
• Experiment 1: Each phage has an insert of approximately 20 kb
• The fact that the cellulase gene is 1 kb not really relevant
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Screening a eukaryotic gene library
• Will the gene express?– No, lack of promoter, ribosome binding
sequence and introns
• Use nucleic acid hybridisation
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1 2 3
4 5 6
7 8 9
1 2 3
4 5 6
7 8 9
1 2 3
4 5 6
7 8 9
NaOH added to lyse bacteriaand denature DNA intosingle strand. Filter is heated to 70 to immobilisesingle strand DNA
Inert DNA to blockvacant DNA binding
sites on filter
Single stranded print ofbacterium’s DNA insteadof a bacterial colony
Plate out E. colicells on nylon filter
Nucleic acid hybridization
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1 2 3
4 5 6
7 8 9
1 2 3
4 5 6
7 8 9
Add radioactivenucleic acid probethat binds to target gene
1 2 3
4 5 6
7 8 9
Expose filter to X-ray filmto detect where nucleic acidhas bound (radioactive andthus blackens X-ray film)
Nucleic acid hybridization
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Screening a eukaryotic gene library
• Where does the probe come from?– Homologous gene from other organism– Oligonucleotide based on protein
sequence or known sequence of homologous gene
– Differential screen (deal with this later; microarray analysis)
![Page 94: Basis of course](https://reader036.fdocuments.in/reader036/viewer/2022062407/56812e5d550346895d9403ce/html5/thumbnails/94.jpg)
Screening a eukaryotic gene library
• Homologous gene from other organism– Mammalian genes are very similar– Thus if trying to get human gene screen with
the equivalent gene from another organism– Oligonucleotide based on protein sequence or
known sequence of homologous gene– Purify protein and determine sequence– Build a nucleotide sequence which codes for
protein sequence
![Page 95: Basis of course](https://reader036.fdocuments.in/reader036/viewer/2022062407/56812e5d550346895d9403ce/html5/thumbnails/95.jpg)
Amino acid sequenceMet-Asn-Lys-Trp-Glu-Met
ATG AAT AAA TGG GAA ATGATG AAT AAA TGG GAG ATGATG AAT AAG TGG GAA ATGATG AAT AAG TGG GAG ATGATG AAC AAA TGG GAA ATGATG AAC AAA TGG GAG ATGATG AAC AAG TGG GAA ATGATG AAC AAG TGG GAG ATG
Met = ATG; Asn = AAT or AAC; Lys = AAA or AAG; Trp = TGG; Glu = GAA or GAG
How many probes must we make?
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1st Test in BNS216
• Test is next Thursday February 26th
• One hour replaces lecture
• Multiple choice and single word or simple diagram answers
• Consists of – Construction and screening of gene
libraries
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Construction of gene libraries
• Gene library– Restriction endonucleases– DNA ligase– Vectors
• Plasmid• Bacteriophage (lambda)• Cosmid• Yeast artificial chromosome
![Page 98: Basis of course](https://reader036.fdocuments.in/reader036/viewer/2022062407/56812e5d550346895d9403ce/html5/thumbnails/98.jpg)
Screening• Bacterial gene library
– Depends on protein expression• Phenotype change e.g. cellulase gene• Complementation of E. coli mutant• Detection of gene product using antibody
• Eukaryotic gene library– No protein expression expected– Nucleic acid hybridisation– Probe
• Homologous gene from other organism• Oligonucleotide probe based on protein sequence
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Cloning cDNA• cDNA: complementary or copy DNA• It is derived from mRNA• double stranded DNA with identical
sequence to mRNA in one strand• Why clone cDNA
– Important in expressing eukaryotic proteins in bacteria
– Introns not removed in bacteria– Therefore not protein produced
![Page 100: Basis of course](https://reader036.fdocuments.in/reader036/viewer/2022062407/56812e5d550346895d9403ce/html5/thumbnails/100.jpg)
Eukaryotic organism
Transcription
RNA
Splicing
Translation
Functionalprotein
Bacteria
Translation
Inactive protein
mRNA
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Bacteria
Transcription
mRNA
Translation
Functionalprotein
cDNA
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Isolate specific cDNA(encodes growth hormone)
• How do isolate mRNA encoding growth hormone
• Isolate cells that express growth hormone– Pituitary
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Isolate specific cDNA(encodes growth hormone)
• Extract mRNA
• Convert to cDNA
• Construct cDNA library
• Screen library for members containing cDNA encoding growth hormone
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Pituitary
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
Isolate total mRNA
Encodes GH
![Page 105: Basis of course](https://reader036.fdocuments.in/reader036/viewer/2022062407/56812e5d550346895d9403ce/html5/thumbnails/105.jpg)
TTTTTT
TTTTTT
Purify mRNA
AAAAAA
AAAAAA
Ribosomal RNA etc
Matrix ispolydTcellulose
Pass total RNA overmatrix. Only mRNAwill bind. Other RNAswill pass directlythrough
AAAAAA
AAAAAA
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TTTTTTAAAAAA
TTTTTTAAAAAA
Low salt buffer
TTTTTT
TTTTTT
AAAAAA
AAAAAA
+
![Page 107: Basis of course](https://reader036.fdocuments.in/reader036/viewer/2022062407/56812e5d550346895d9403ce/html5/thumbnails/107.jpg)
cDNA synthesis• Use three enzymes
– reverse transcriptase– RNAase H– DNA polymerase
• Reverse transcriptase– Converts RNA into a complementary DNA sequence– Add onto existing double stranded nucleic acid– Can’t initiate DNA synthesis– Use primer to initiate DNA synthesis
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5’ 3’
5’ 3’
5’ 3’
5’ 3’3’ 5’
mRNA
primer
Reversetranscriptase
![Page 109: Basis of course](https://reader036.fdocuments.in/reader036/viewer/2022062407/56812e5d550346895d9403ce/html5/thumbnails/109.jpg)
Primer
• Small single stranded DNA sequence
• Binds to 3’ end of all mRNA
• What is the sequence?– PolydT
![Page 110: Basis of course](https://reader036.fdocuments.in/reader036/viewer/2022062407/56812e5d550346895d9403ce/html5/thumbnails/110.jpg)
AAAAAATTTTTT
5’ 3’
AAAAAATTTTTT
5’ 3’
3’5’
AAAAAATTTTTT
5’ 3’
3’5’
AAAAAATTTTTT
5’ 3’
3’5’
AAAAAATTTTTT
5’ 3’
3’5’
AAAAAA 5’ 3’
TTTTT Add primer
Reverse transcriptase + dNTPs
mRNAcDNA
mRNA
mRNA
RNAase H
DNA polymerase + dNTPs
Completed DNA synthesis
Double stranded cDNA
![Page 111: Basis of course](https://reader036.fdocuments.in/reader036/viewer/2022062407/56812e5d550346895d9403ce/html5/thumbnails/111.jpg)
Collection of cDNA molecules
• Insert cDNAs into a vector– Generate recombinant vectors
• Insert recombinant vectors into a bacterium such as Escherichia coli
• This comprises a cDNA library derived from pituitary mRNA
• Screen cDNA library for those containing growth hormone encoding cDNA
![Page 112: Basis of course](https://reader036.fdocuments.in/reader036/viewer/2022062407/56812e5d550346895d9403ce/html5/thumbnails/112.jpg)
cDNA
GAATTCCTTAAG
GAATTCCTTAAG
GAATTCCTTAAG
Add linkers at high concentrationsand DNA ligase
GCTTAAG
AATTC G
Cut with EcoRI
Insert into a vectorcut with same enzymefollowed by DNA ligase
cDNA is blunt-ended thus ligatesto other DNA unless concentration high
![Page 113: Basis of course](https://reader036.fdocuments.in/reader036/viewer/2022062407/56812e5d550346895d9403ce/html5/thumbnails/113.jpg)
Linkers
• Small complementary oligosaccharides– Synthesised in large amounts
• Contain sequence cleaved with a restriction enzyme• Blunt ended double strand molecules• Because synthesised at high concentrations can be
ligated efficiently to other double stranded molecules (cDNA molecules)
• Once ligated to cDNA cut with restriction enzyme to create sticky ends
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cDNA
GAATTCCTTAAG
GAATTCCTTAAG
GAATTCCTTAAG
Add linkers at high concentrationsand DNA ligase
GCTTAAG
AATTC G
Cut with EcoRI
Insert into a vectorcut with same enzymefollowed by DNA ligase
cDNA is blunt-ended thus ligatesto other DNA unless concentration high
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Transform intoE. coli
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Screen for members oflibrary that containgrowth hormone cDNA