Recombinant Technology by HC120914111156

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									Construction of Recombinant
Microbe
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
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.
Recombinant DNA

    The combination of fragments
     of DNA from different
     sources.

     (Cutting and pasting DNA
      fragments together)
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)
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.
Sticky ends

 When the ends of the
  restriction fragments
  are complementary,
 EcoRI – recognition
  sequence

    5'---G ‘ AATTC---3'
     3'---CTTAA ‘G---5'
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
Pasting DNA

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


                 Complementary
                  ends (sticky ends)
                  H-bond

                 Ligase forms
                  phosphodiester
                  bond to seal
                  strands together.
Restriction enzymes generate fragments
     that facilitate recombination
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
Experimental Design
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
pGlo (an example of plasmid vector)

    Ori
    Polylinker cloning
     region
    Amp (beta lactamase
     for resistance)
    araC( arabinose operon)
    pBad promoter
    Green fluorescent
     protein - reporter
Other desirable properties of
Vector
 High copy number
 Inducible promoter under stringent
  control
 Stable incorporation (especially for
  improvement of microbial traits)
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
Control of expression using the
lcI repressor
pET expression in E. coli
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
Purification of recombinant fusion
proteins expressed in E. coli
Properties of Host (especially for
production of recombinant protein)

   Rapid growth
   Cheap substrates
   Not fastidious
   Low toxicity/pathogenicity
     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
     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
    Expression hosts (bacteria)

   Streptomyces
       Well understood fermentation, difficult
        transformation, moderate-slow growth, not
        fastidious, extracellular protein, high yields,
        limited range of vectors
    Expression hosts (yeast)

   Saccharomyces
       Very well understood fermentation, difficult
        transformation, fast growth, not fastidious,
        extracellular protein, high yields, limited range
        of vectors
    Expression hosts (fungi)

   Trichoderma
       Poorly understood fermentation, difficult
        transformation, slow growth, not fastidious,
        extracellular protein, high yields, limited range
        of vectors
  How to put plasmid into an E. coli
  cell?
ORI                                                                                                        Cells that do not
              Amp R.
                                                                                                          take up plasmids
                                     ORI               Amp R.                                              die on ampicillin
                                                                                                                plates


  Plasmid vector
                    Enzymatically                        Mix E.coli cells with
                   insert DNA into                     plasmids in presence of
      +             plasmid vector
                                           Recombinant
                                             plasmid   CaCl2 Culture on nutrient
                                                                                                           Transformed
                                                                                                            E.coli cell
                                                        agar plates containing                               survives
  DNA fragment                                                ampicillin
   to be cloned                                                                     Bacterial
                                                                                  chromosome

                                                                                                Independent
                                                                                                  plasmid
                                                                                                 replication
                                                                      Cell
                                                                 multiplication
Transformation

 Putting a plasmid (a vector of a
 vector carrying an inserted gene)
 inside a host cell
Transformation (1)
    PRE-INCUBATION
     The 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.
Transformation (2)
    * INCUBATION
     The 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.
Transformation (3)

    * HEAT SHOCK
     The 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.
Colonies of E. coli carrying pGlo
E. coli carrying pGlo
Recombinant DNA

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

      Viral and human
Uses of Recombinant Microbes




                     control
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
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
Uses of Recombinant Microbes

 Production    of commercial protein
 products
   Therapeutic   proteins
     Insulinfor diabetes treatment
     Interferon-gamma for cancer
      treatment
     Factor VIII

     Erythropoetin

     Epidermal growth factor
Example: Production of
recombinant human insulin in E. coli
            Many diabetic patients need to administer
            insulin to control their blood glucose levels




                                                      Lilly
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

     N
      B chain
     A chain C
      C chain
 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_rdna_insulin.htm
 Example: Production of
 recombinant human insulin in E. coli
  Induce lac expression
                                     Mix purified insulin chains A and B


                                     Refold under oxidising conditions to
                                     promote disulphide bond formation




Purification and cleavage of b-gal
  protein tag from insulin chain
                                                 Humulin
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
Integration Vector

   Plasmid



   Chromosome
            legume




Fixed nitrogen     Fixed carbon
  (ammonia)      (malate, sucrose)




            rhizobia
             Biological nitrogen fixation:

                                                             nitrogenase
N2 + 8   flavodoxin-   +   8H+   + 16   MgATP2-   + 18 H2O
  2NH4+ + 2OH- + 8 flavodoxin + 16 MgADP- + 16H2PO4- + H2



     1. Rare, extremely energy consuming conversion
        because of stability of triply bonded N2
     2. Produces fixed N which can be directly
        assimilated into N containing biomolecules
Genes involved in N2-fixation
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 KNO3–2 mMNH4NO3.
The use of microbe for
plant genetic engineering
Agrobacterium tumefaciens and
nature’s genetic engineering
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
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
Ti plasmid
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
Genetic Engineering and Ti
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
See you ….

								
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