Construction of Recombinant Microbe

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Construction of Recombinant Microbe
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What is Recombinant Microbe

• A microbe which is genetically modified by
  applying DNA Recombinant Technology
• A microbe which acquires foreign gene through DNA
  Recombinant Technology
• An organism is called transgenic if it has
  genetic information added to it from different
  type of organism

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Recombinant DNA technology

• A DNA technology that utilizes the power of
  microbiological selection and screening
  procedures to allow investigators to isolate a
  gene that represents as little as 1 part in a
  million of the genetic material in an organism.

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Recombinant DNA
The combination of fragments of DNA from
different sources. (Cutting and pasting DNA
fragments together)
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Cutting DNA using Restriction enzymes

• Restriction Enzymes
• Isolated from various bacteria, restriction
  enzymes recognize short DNA sequences and cut the
  DNA molecules at those specific sites.
• (A natural biological function of these enzymes
  is to protect bacteria by attacking viral and
  other foreign DNA)

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Process

• Restriction endonucleases cut at defined
  sequences (palindromic) of (usually) 4 or 6 bp.
  They cut on both strands of DNA.
• This allows the DNA of interest to be cut at
  specific locations.
• Cuts yield either "sticky" ends, or "blunt" ends.

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Sticky ends

• When the ends of the restriction fragments are
  complementary,
• EcoRI recognition sequence
• 5'---G AATTC---3'
• 3'---CTTAA G---5'

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Blunt ends

• When the restriction endonuclease cleaves in the
  center of the pseudopalindromic recognition site
  to generate blunt (or flush) ends.
•   HaeIII GG CC
• CC GG

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Pasting DNA

• Two pieces of DNA cut with the same enzyme, can
  be pasted together using another enzyme called
  "DNA ligase".

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Pasting DNA

• Complementary ends (sticky ends) H-bond
• Ligase forms phosphodiester bond to seal strands
  together.

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Restriction enzymes generate fragments that
facilitate recombination
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Process for Recombinating DNA

• Cut ends in recognition sequence
• Open DNA
• Recombine with another piece of DNA cut with the
  same restriction enzyme
• Use ligase to seal the cuts and rejoin the
  fragments

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Experimental Design
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Plasmid Vectors

• Ori (origin of replication)
• Polylinker cloning sites
• Regulatory region (lac operon)
• Antibiotic resistance gene(s)
• Reporter gene for protein color or fluorescent
  molecule

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pGlo (an example of plasmid vector)

• Ori
• Polylinker cloning region
• Amp (beta lactamase for resistance)
• araC( arabinose operon)
• pBad promoter
• Green fluorescent protein - reporter

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Other desirable properties of Vector

• High copy number
• Inducible promoter under stringent control
• Stable incorporation (especially for improvement
  of microbial traits)

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Regulating protein expression in E. coli

• Expression often deleteriously affects growth of
  the host cell
• Therefore, expression is usually tightly
  regulated using specific promoter constructs
• Expression is the divided into two main phases
• Cell growth phase (biomass generation) -
  expression switched off
• Expression phase - expression induced

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Control of expression using the lcI repressor
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pET expression in E. coli
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E. coli expression vector

• pUC features
• ori
• AmpR
• multiple cloning sites
• T7 SP6 promoters
• Regulated expression
• tac promoter (fusion between trp and lac
  promoters)
• regulated like lac (IPTG)
• Fusion protein
• purification tag
• cleavable with Xa protease

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Purification of recombinant fusion proteins
expressed in E. coli
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Properties of Host (especially for production of
recombinant protein)

• Rapid growth
• Cheap substrates
• Not fastidious
• Low toxicity/pathogenicity

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Expression hosts (bacteria)

• E. coli
• Very well understood genetics and fermentation,
  rapid growth, not fastidious, wide range of
  vector systems, very easy transformation,
  intracellular protein, low yields

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Expression hosts (bacteria)

• Bacillus
• Very well understood genetics and fermentation,
  difficult transformation, very rapid growth, not
  fastidious, intracellular protein, high yields,
  limited range of vectors

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Expression hosts (bacteria)

• Streptomyces
• Well understood fermentation, difficult
  transformation, moderate-slow growth, not
  fastidious, extracellular protein, high yields,
  limited range of vectors

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Expression hosts (yeast)

• Saccharomyces
• Very well understood fermentation, difficult
  transformation, fast growth, not fastidious,
  extracellular protein, high yields, limited range
  of vectors

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Expression hosts (fungi)

• Trichoderma
• Poorly understood fermentation, difficult
  transformation, slow growth, not fastidious,
  extracellular protein, high yields, limited range
  of vectors

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How to put plasmid into an E. coli cell?
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Transformation

• Putting a plasmid (a vector of a vector carrying
  an inserted gene) inside a host cell

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Transformation (1)

• PRE-INCUBATIONThe recipient E. coli cells will
  be exposed to positively charged calcium chloride
  (CaCl2) ions. This treatment is meant to stress
  the bacterium in order to render its cell
  membrane and cell wall permeable to the donar
  plasmid. This process will make the recipient E.
  coli "competent" to uptake the plasmid.

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Transformation (2)

• INCUBATIONThe plasmid (with amp gene) is
  added to a recipient E. coli suspension, which
  will now be called E. coli because it is the
  one which is being transformed. Another E. coli
  suspension will act as a control, called E. coli
  - because it will not be exposed to the plasmid
  therefore, it will NOT inherit the gene.

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Transformation (3)

• HEAT SHOCKThe recipient cells plus plasmids
  and the control cells not exposed to the plasmids
  are briefly exposed to 42 degrees C. This step
  will maximize the uptake of the plasmid through
  the wall and membrane of the cells.

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Colonies of E. coli carrying pGlo
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E. coli carrying pGlo
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Recombinant DNA

• Combination of DNA from organisms from two
  different sources
• Bacterial and human
• Bacterial and plant
• Viral and human

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Uses of Recombinant Microbes
control
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Uses of Recombinant Microbes

• 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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Uses of Recombinant Microbes

• 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

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Uses of Recombinant Microbes

• Production of commercial protein products
• Therapeutic proteins
• Insulin for diabetes treatment
• Interferon-gamma for cancer treatment
• Factor VIII
• Erythropoetin
• Epidermal growth factor

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Example Production of recombinant human insulin
in E. coli
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Example Production of recombinant human insulin
in E. coli

• Insulin is synthesized in pancreatic islet
  cells.
• It is made as a single polypeptide chain
• preproinsulin.
• Preproinsulin is proteolytically processed to
• form Insulin
• In mature Insulin, the A and B chains are
  linked
• by disulphide bridges

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Example Production of recombinant human insulin
in E. coli
Synthetic Insulin Chain A and Chain B sequences
cloned separately into a lac-based expression
vector
http//members.tripod.com/diabetics_world/lillys_r
dna_insulin.htm
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Example Production of recombinant human insulin
in E. coli
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Uses of Recombinant Microbes

• Improvement of microbial traits
• Increasing N2 fixation ability
• Ability to use complex substrate such as,
  cellulose, xylose, and amylum
• Resistance to drought, heavy metal other toxic
  compounds

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Integration Vector
Plasmid
Chromosome
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(No Transcript)
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Biological nitrogen fixation
nitrogenase
N2 8 flavodoxin- 8H 16 MgATP2- 18 H2O
2OH- 8 flavodoxin 16 MgADP- 16H2PO4- H2
2NH4

• Rare, extremely energy consuming conversion
  because of stability of triply bonded N2
• Produces fixed N which can be directly
  assimilated into N containing biomolecules

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Genes involved in N2-fixation
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Effect of nifH overexpression on nitrogen
fixation and plant growth
Growth response of P. vulgaris plants (45 dpi)
inoculated with R. etli strains in the
greenhouse. Images 1, Noninoculated
nonfertilized 2, inoculated with CFN42 (wt) 3,
inoculated with HP55 (nifHcDK) 4, noninoculated
fertilized with 10 mM KNO32 mMNH4NO3.
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The use of microbe for plant genetic engineering
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Agrobacterium tumefaciens and natures genetic
engineering
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Nature of the Microbe

• A. tumefaciens is a Gram-negative, non-sporing,
  motile, rod-shaped bacterium,
• Closely related to Rhizobium which forms
  nitrogen-fixing nodules on clover and other
  leguminous plants.
• Possesses a large, natural plasmid called Ti

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Agrobacterium tumefaciens

• Attracted to wounds or openings in the plant cell
  wall
• Uses acetosyringone to inject into the plant
  cells
• Ti plasmid enters the plant cell and integrates
  randomly into the host
• Plasmid codes or opines and nopalines two
  distinctive gene products that lead to tumor
  production in infected plants

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Ti plasmid
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Ti plasmid and genes

• ori--replication controlled sites
• tra region--responsible for mobility from
  bacteria to plant cell
• vir--induce uncontrolled cell division in the
  host plant
• t region (tDNA)--group of genes that control the
  transfer of the tDNA to the host chromosome

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Genetic Engineering and Ti
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Uses of Recombinant Microbes

• For environmental applications
• Oil eating microbes Prince William Sound
  Alaska
• Degradation of mercury in the environment Clean
  up of contaminated sites

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See you .