Department of Biotechnology, University of the Western Cape MIC231 Molecular and Environmental Micro

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Department of Biotechnology, University of the
Western CapeMIC231 Molecular and Environmental
Microbiology

• Lecture 6-8 rDNA technology

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Essay

• Describe and discuss microbial genome sequencing.
  
• Essays, which should be between 1500 and 3000
  words, should be handed in (or submitted
  electronically) to Professor Cowan, room 128
  (dcowan_at_uwc.ac.za) by 1700h on Monday 16th
  September.
• Feel free to use www resources
• Plagiarism (copying/downloading etc) of written
  material will not be acceptable.
• Hand-written essays will not be accepted.
• A penalty of 20 per week will apply to late
  submissions.

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Lectures 6 - 8

• 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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Structure of nucleic acid backbone
5-hydroxyl
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4
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2
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3-hydroxyl
-H in DNA, -OH in RNA
Phosphodiester bond
Exist as right-handed double helices (DNA) or
single stranded RNA. The helix is stabilised by
base-pairing (G C) and (AT).

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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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Cloning prokaryotic and eukaryotic genes

• Prokaryotes (Bacteria and Archaea) contain
  complete and uninterrupted ORFs - therefore
  prokaryote genes can be cloned directly from
  genomic DNA
• All higher (and many lower 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

• 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

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Prokaryote gene cloning strategies 1. Shotgun
cloning
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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DNA extraction

• 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.
• Lysozyme treatment
• Lysozyme is an enzyme which degrades cell wall
  peptidoglycans, causing cells to become weakened,
  and subject to osmotic lysis. Detergents help to
  dissolve membranes and denature proteins.
• DNA can be further purified by several methods
  including phenol-chloroform treatment, CsCl
  gradient centrifugation and ion exchange
  chromatography.
• These methods can be used to isolate both
  chromosomal and plasmid DNA.

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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, and 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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Some restriction enzymes and their cut sites
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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
Mobility (mm)
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DNA ligation and Vectors

• DNA ligases use ATP to join phosphodiester bonds
  in annealed DNA (i.e., where cohesive sticky
  ends occur)
• DNA fragments can be ligated into a vector IF the
  vector is cut with the same RE as the restricted
  DNA.
• It is important that the vector DNA is cut with
  an enzyme have 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
-G A-T-C- -C-T-A G-
DNA ligase ATP
-G-A-T-C- -C-T-A-G-
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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) or
  infrequently (low copy number)
• Plasmids are widely used as cloning vehicles
• Important components of plasmids are
• 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 insert-containing
  plasmids

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Diagram of a typical plasmid
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Transforming microbial host cells

• E. coli is the commonest cloning host.
• E. coli can be induced to accept plasmid DNA.
• Common E. coli-specific plasmids are pBR322 (4363
  bp) and pUC19 (2686 bp) many sophisticated
  commercial vector systems have been developed
  from these basic plasmids.
• E. coli can be transformed with plasmid DNA
  (i.e., induced to take up plasmid DNA) by several
  methods which make the cell wall/membrane
  temporarily leaky
• CaCl2 treatment
• Polyethylene glycol
• Electroporation
• Typically, a single E. coli cell will accept
  only one plasmid molecule.

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Diagram of cells, plasmids and transformation 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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Plating out a library and selecting clones

• Libraries are plated on media containing
  antibiotics (e.g., Ampicillin).
• Only colonies containing plasmids with the AmpR
  gene will grow deletes un-transformed clones
• Blue-white selection is used to identify colonies
  which have insert-containing plasmids can
  ignore those that have plasmids with no DNA
  insert
• The blue-white selection involves addition of
  ITPG and X-gal to the medium
• ITPG induces the LacZ operon
• 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
  (remember that the MCS is 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

• 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.
• 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.
• 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 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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Example of complementation screening

• 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.
• Any cell which grows MUST have been
  complemented in the missing gene i.e., the
  plasmid in that clone MUST contain the missing
  Leu biosynthesis gene.

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Example of hybridisation screening

• Hybridisation involves the binding of a single
  stranded DNA sequence to a complementary ssDNA
  sequence note non-complementary sequences will
  not bind.
• This process is often known as Southern Blotting
• 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 an 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 Southern Blotting process
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Prokaryotic gene cloning. 2. PCR cloning

• Design PCR primers which are complementary to
  regions of the gene
• 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
• Amplify a partial gene sequence from genomic DNA
  using the polymerase chain reaction
• 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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Notes of the Polymerase Chain Reaction

• 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
• 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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Applications of rDNA technology

• Production of protein for analytical and
  structural analysis
• Native and mutant proteins for functional
  analysis
• Protein for structural (e.g., x-ray
  crystallographic) analysis
• 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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Requirements of industrial production of
recombinant proteins

• 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. 2.

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

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Expression hosts

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