Department of Biotechnology, UWC MICR321

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BTY 227Molecular and Environmental
BiotechnologyLectures 5-7Recombinant DNA
Technology
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Overview

• Cloning strategies introduction
• Cloning prokaryote and eukaryote genes
• Preparation of DNA
• Vectors
• DNA-manipulating enzymes
• Cloning pathways
• Cell transformation
• Expression and selection strategies
• Industrial applications of rDNA technology

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Gene expression
To the ribosome for protein synthesis
(translation)
mRNA
Promoter sequence
ATG Open Reading Frame
TAA Start Stop Codon Codon
Constitutive expression. RNA polymerase binds to
the promoter region, synthesises mRNA for
subsequent translation into protein.
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What is gene cloning?

• Making multiple copies of a single gene.
• Finding the target gene in chromosomal DNA
• Cutting out the full-length gene as a DNA
  fragment
• Ligating the gene into a suitable vector (a
  vector is a transporter of DNA from one
  organism to another)
• Inserting the vector into a host cell
• Amplifying the cell numbers
• Fig 1

Essential reading Prescott, 5th Edn., pages
319-343
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Cloning pro and eukaryotic genes

• Prokaryotes contain complete and uninterrupted
  ORFs - therefore prokaryote genes can be cloned
  directly from genomic DNA
• Most eukaryotes have ORFs which are divided into
  coding (exon) and non-coding (intron) sequences
  therefore these genes cannot be cloned directly
  from genomic DNA - these require complementary
  DNA (cDNA) cloning.
• Some lower eukaryotes (e.g., Saccharomyces) have
  a mixture of intron-free and intron-containing
  genes.

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Notes on cDNA cloning for eukaryotic genes

• Isolate mRNA (the intronic sequences have been
  spliced out by the cell in the synthesis of mRNA)
• Reverse transcribe mRNA to generate cDNA (using
  an RNA-virus enzyme called Reverse
  Transcriptase).
• Clone as for genomic DNA
• Fig 2

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Shotgun cloning a prokaryote gene cloning
strategy
Lyse cells and extract DNA
Restrict DNA
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1
Genome
Target gene
Ligate into plasmid vector cut with the same
restriction enzyme
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Colony containing target gene
Plate library on selective media
Transform host cells
A population of plasmids where each plasmid
contains a different DNA fragment
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A population of host cells, where, each cell
contains a plasmid with a different DNA fragment
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Extracting DNA from cells

• Cell lysis (breaking the cell open)
• Bead-beating - a technique where shaking cells
  in the presence of small silica beads (20
  200microns) breaks cell walls. Detergents help to
  dissolve lipids and denature proteins. A vigorous
  method which can shear DNA.
• EDTA/Lysozyme treatment Lysozyme is an enzyme
  which degrades cell wall peptidoglycans, causing
  cells to become weakened, and subject to osmotic
  lysis. Detergents (SDS) help to dissolve
  membranes and denature proteins.

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Extracting DNA from cells (2)

• DNA can be further purified by several methods
  including phenol-chloroform treatment, CsCl
  gradient centrifugation and ion exchange
  chromatography
• Fig 3

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Preparation of plasmid DNA (1)

• Methods based on differences in size or
  conformation of plasmid and chromosomal DNA
• Size
• chromosomes much larger than plasmids
• If lysis is gentle, little breakage of chromosome
  will occur
• Also, chromosome is physically attached to cell
  envelope
• When centrifuged, chromosomal DNA will sediment
  with cell debris while plasmid will remain in
  supernatant Fig 4

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Preparation of plasmid DNA (2)

• Separation based on conformation
• Plasmids usually exist as supercoiled (ccc)
  molecules
• If one of the strands is nicked, the plasmid
  relaxes and is in its open circular state Fig 5
• Supercoiled plasmids are easily separated from
  open circular and linear fragments of DNA by the
  alkaline denaturation technique
• Fig 6

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DNA restriction

• Restriction endonucleases (restriction enzymes)
  cleave the phosphodiester bonds of double
  stranded DNA, creating double stranded breaks
• Restriction enzymes recognise palindromic
  sequences in DNA sequence with a two-fold
  symmetry,
• Some make staggered cuts, with sticky ends.

GAATTC G
AATTC CTTAAG CTTAA
G
Cleavage of dsDNA by EcoR1
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Restriction enzymes

• There are hundreds of different restriction
  enzymes with unique recognition sequences and cut
  sites
• REs are named after the microorganisms from
  which they are produced EcoR1
• EcoR1 E. coli R1
• HinDIII Haemophilus influenzae DIII
• Sau3A Staphylococcus aureus 3A
• Recognition sites differ in length and sequence
• The length of the recognition sequence dictates
  how often the RE will cut a piece of DNA

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Restriction enzymes and their cut sites
Fig 7
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Separation and sizing of DNA fragments

• Agarose gel electrophoresis is used for DNA
  separation
• Effective for fragments between 0.2 kb and 20kb

-ve
23 9.4 6.6 4.4 2.3 2.0 0.56

• lHinDIII ladder
• pBR322 plasmid
• pBR322 cut
• with Acc1.

Direction of mobility
ve
1 2 3
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Calculating fragment sizes

• For marker fragments, measure distance of each
  fragment from top of the gel
• Plot mobility vs log mw (bp)
• Determine unknown sizes from standard curve

Log mw
Estimate size of unknown fragment
Mobility of unknown fragment
Fig 8
Mobility (mm)
Figure 8
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Plasmid vectors

• Plasmids are circular ds DNA units which
  replicate autonomously in bacteria
• Plasmids vary widely in size (lt1kb - gt 50kb)
• Plasmids may replicate frequently (multicopy
  50-100) or infrequently (low copy number 2-10)
• Plasmids are widely used as cloning vehicles

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Diagram of a typical plasmid
Multiple cloning site, positioned inside the
lacZ gene(pUC8) or the Tet resistance gene
(pBR322)
Antibiotic resistance Gene e g amp
ori (origin of replication) sequence
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Important components of plasmids vectors

• Ori sequence origin or replication determines
  copy number
• Multiple cloning site multiple unique
  restriction sites for cloning
• Antibiotic resistance marker(s) only host cells
  containing the vector will grow
• LacZ insertional inactivation sequence basis of
  blue-white screening makes it possible to
  determine which clones contain recombinant
  plasmids

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DNA ligation and vectors

• DNA fragments can be ligated into a vector IF the
  vector is cut with the same RE as the restricted
  DNA.
• DNA ligases use ATP to join phosphodiester bonds
  in annealed DNA (i.e., where cohesive sticky
  ends occur)
• It is important that the vector DNA is cut with
  an enzyme having a single restriction site in the
  vector (i.e., the vector is linearised, not
  fragmented).
• All cloning vectors have been redesigned to have
  a multiple cloning site (MCS) which has a
  sequence of unique restriction sites.

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The ligation reaction
-G A-T-C- -C-T-A G-
Sticky ends anneal
Note The strands are not covalently joined
-G A-T-C- -C-T-A G-
DNA ligase ATP
-G-A-T-C- -C-T-A-G-
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Products of ligation

• Following ligation, the ligation mixture may
  contain the following
• The desired recombinant molecule
• Unligated vector molecules
• Unligated DNA fragments
• Self ligated (religated) vector molecules
• Recombinant molecules that contain the wrong
  (undesired) insert DNA
• Fig 9

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Transformation of host cells

• E. coli is the commonest cloning host.
• E. coli can be induced to accept plasmid DNA
  (made competent)
• Common E. coli-specific plasmids are pBR322 (4363
  bp) and pUC19 (2686 bp)
• Typically, a single E. coli cell will accept only
  one plasmid molecule.

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Transformation methods

• E. coli can be transformed with plasmid DNA by
  several methods which make the cell wall/membrane
  temporarily leaky
• CaCl2 treatment
• Polyethylene glycol
• Electroporation

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Diagram of cells, plasmids and transformation a
plasmid library!
Treat cells to make them competent
Mix competent cells and plasmids
Host cells
Plasmid vectors
A plasmid library
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Calculating library sizes

• For a single microbial genome, size is typically
  4-8Mbp
• For average plasmid insert size of 1.5kb, would
  require a library of 3000 - 6000 clones to
  represent a complete genome.
• With a complete digestion, many ORFs will be
  cleaved internally.
• To generate a library with complete ORFs, clone
  larger fragments, or perform partial digest
  (resulting in larger library).

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Library plating and clone selection

• Libraries are plated on media containing
  antibiotics (e.g., Ampicillin).
• Only colonies containing plasmids with the AmpR
  gene will grow this eliminates all
  un-transformed clones
• Fig 9

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Blue white selection (1)

• Blue-white selection is used to identify colonies
  which have insert-containing plasmids
  eliminates those that have plasmids with no DNA
  insert
• Use a pUC cloning vectors and host cells which
  produce an incomplete (inactive) b-galactosidase
  protein
• MCS in pUC vector is in the is in the middle of
  the LacZ gene
• The blue-white selection involves addition of
  ITPG and X-gal to the medium
• ITPG induces the LacZ operon
• Fig10 and 11

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Blue white selection (2)

• The LacZ operon results in expression of the
  b-galactosidase a-peptide
• The b-galactosidase a-peptide complements the
  incomplete (inactive) b-galactosidase protein in
  the host E. coli cells and produces functional ,
  active b-galactosidase
• Functional b-galactosidase cleaves the colourless
  X-gal in the medium to give active colonies a
  blue colour.
• IF a plasmid contains a DNA insert in the MCS
  (i.e. in the middle of the LacZ gene), then a
  functional a-peptide cannot be generated,
  complementation does not occur, and colonies
  cannot cleave X-gal. Therefore, colonies with
  plasmids with inserts stay white.

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Other targeted library screening options

• We discuss 3 methods
• Activity detection
• Complementation screening
• Hybridisation (Southern blotting)

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Activity detection (expression screening)

• Library is plated on media containing a substrate
  for the target gene product (e.g., an enzyme
  substrate). A physical change occurring when the
  enzyme reacts with the substrate, such as a
  colour change, indicates that the gene is
  expressed.

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Example of activity detection

• Detection of cloned alpha-amylase genes
• Clones are plated on an agar medium containing
  starch
• Plates are incubated to allow single cells to
  develop into colonies
• Clones expressing a-amylase genes will hydrolyse
  the starch in the vicinity of the colony
• Plates are flooded with iodine/KI (which stains
  starch blue)
• Colonies with a clear halo around them are
  expressing a-amylase.

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Detection of amylase-producing E. coli clones
using a starch-iodine/KI expression detection
system
Clearing zone indicates starch hydrolysis
i.e., amylase-producing clone
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Complementation screening

• The library is plated on a medium lacking a
  critical component for growth. Only those
  colonies expressing a gene capable of producing
  that component will grow.
• Example shown is screening for leucine
  biosynthesis genes

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• Leucine biosynthesis genes
• Transform plasmid DNA into auxotrophic E. coli
  mutant (an auxotrophic mutant is one which cannot
  synthesise a critical cell component - such as
  the amino acid leucine - and requires that
  component to be added in the medium before it can
  grow. Leu-minus auxotrophic mutants lack one of
  the key Leu biosynthesis genes.
• Spread E. coli library on agar medium containing
  C and N nutrient sources, but deficient in Leu.
• Cells which grow MUST be complemented in the
  missing gene i.e., the plasmid in that clone
  MUST contain the missing Leu biosynthesis gene.

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Hybridisation (Southern blotting)

• The presence of a target gene is detected by
  hybrisidation with a complementary gene sequence,
  linked to a reporter (radioactive marker,
  enzyme-linked marker, GFP)

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Example hybridisation screening

• Hybridisation involves the binding of a single
  stranded DNA sequence to a complementary ssDNA
  sequence note non-complementary sequences will
  not bind.
• It is possible to identify the presence of a
  complementary sequence on an agarose gel or in a
  colony by Southern Blotting with a hybridisation
  probe.

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Southern Blotting on agarose gel

• Extract plasmid DNA from a clone,
• Electrophorese on an agarose gel
• Transfer DNA to a nylon membrane (blotting)
• Treat DNA to make single stranded
• Wash membrane with hybridisation probe (a single
  stranded piece of DNA), labeled so that it can be
  detected.
• Wash membrane to remove unbound probe
• Apply detection method
• Enzyme-linked assay for enzyme-labeled probe
• Radioactive detection for 32P-radioactively
  labeled probe

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Diagram of a Southern Blot
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PCR cloning (1)

• The second prokaryotic gene cloning strategy we
  will discuss (shotgun cloning was the first)
• Design PCR primers which are complementary to
  regions of the gene of interest. How?
• By purifying the protein, obtaining N-terminal
  and/or internal amino acid sequence data, and
  designing the nucleotide sequence from codon
  usage information, or
• By computationally aligning known gene sequences
  and identifying regions of sequence conservation

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PCR cloning (2)

• Amplify a partial gene sequence from genomic DNA
  using the polymerase chain reaction (see next
  slide)
• Purifying the PCR amplicons (sequence to check
  its the right gene!)
• Label the amplicon sequences and use as southern
  Blotting probe to identify the full-length gene
  in a genomic library (see earlier).

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Polymerase Chain Reaction (1)

• PCR has revolutionised molecular biology!
• The success of the method is based on the
  properties of the DNA polymerase enzyme which
  adds complementary nucleotides to ssDNA to form a
  complementary second strand.
• DNA polymerase is primed from a short
  complementary sequence (typically an 18-22-mer)
• In the presence of cofactors and the four
  deoxynucleotides (dnTPs), the enzymes reads along
  the ss template building a complementary strand

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Polymerase Chain Reaction (2)

• dsDNA can be PCR-amplified by using forward and
  reverse primers, complementary to both forward
  and reverse strands.
• The real secret of the success of PCR is the
  ability to cycle the process in an exponential
  amplification i.e., 2 strands become 4, and 8,
  and 16, and 32, and 64.!
• The cycling is made possible by the use of a
  thermostable DNA polymerase (Taq, Pfu, Vent)
  which can withstand the temperature changes
  imposed for the successive cycles of strand
  melting, primer binding and elongation (94oC,
  52oC, 72oC).

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Two limitations of PCR

• PCR experiment can be completed in less than 2
  hours whereas it takes weeks to clone a gene?
• PCR primers can only be designed for genes that
  have been studied before thus if a gene hasnt
  been studied before, cloning might be the only
  option
• There is a limit (/-5kB) to the length of DNA
  sequence that can be copied by PCR. This is
  shorter than the length of many genes

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Applications of rDNA technology

• Look at
• Production of protein for analytical and
  structural analysis
• Production of commercial protein products
• Industrial enzymes
• Therapeutic proteins

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Production of protein for analytical and
structural analysis

• Native and mutant proteins for functional
  analysis
• Protein for structural (e.g., x-ray
  crystallographic) analysis

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Production of commercial protein products

• Industrial enzymes
• Amylase, amyloglucosidase and xylose isomerase
  for the starch industry
• Proteases, cellulases and lipases for the
  detergents industry
• Proteases for the cheese industry
• Penicillin acylase for the pharmaceutical
  industry
• Therapeutic proteins
• Insulin for diabetes treatment
• Interferon-gamma for cancer treatment

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Industrial production of recombinant proteins -
requirements

• Vector
• High copy number
• Inducible promoter under stringent control
• Stable incorporation
• Host
• Rapid growth
• Cheap substrates
• Not fastidious
• Low toxicity/pathogenicity

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Industrial production of proteins.

• Fermentation system
• Easy to control
• Easily scaleable
• Down-stream processing
• Easy removal of cells
• Extracellular product
• Overall requirments
• Cheap operation
• Safe operation
• rProtein production at gram/litre
• Production cost of 5-20/kg

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Expression hosts

• Commonly used host cells for recombinant protein
  production are
• E. coli
• Bacillus
• Streptomyces
• Trichoderma
• Saccharomyces
• Insect, animal and plant cells

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E. coli, Bacillus and Streptomyces

• E. coli
• Well understood genetics and fermentation, rapid
  growth, not fastidious, wide range of vector
  systems, easy transformation, intracellular
  protein, low yields
• Bacillus
• Well understood genetics and fermentation,
  difficult transformation, rapid growth, not
  fastidious, intracellular protein, high yields,
  few vectors
• Streptomyces
• Well understood fermentation, difficult
  transformation, moderate-slow growth, not
  fastidious, extracellular protein, high yields,
  few vectors

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Trichoderma, Saccharomyces and other cells

• Trichoderma
• Poorly understood fermentation, difficult
  transformation, slow growth, not fastidious,
  extracellular protein, high yields, limited range
  of vectors
• Saccharomyces
• Very well understood fermentation, difficult
  transformation, fast growth, not fastidious,
  extracellular protein, high yields, limited range
  of vectors
• Insect, animal and plant cells
• Very poorly understood and difficult
  fermentations, very difficult transformation,
  slow growth, very fastidious, intracellular
  protein, low yields, glycosylated protein products