Recombinant DNA, Biotechnology, and Microbes

1
Recombinant DNA, Biotechnology, and Microbes

• Microbiology 221

2
Overview Putting microbes to Work Molecular
Cloning

• Recombinant DNA technology 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.
• The DNA from the organism of interest is divided
  into small pieces that are then placed into
  individual cells (usually bacterial).
• These can then be separated as individual
  colonies on plates, and they can be screened
  through rapidly to find the gene of interest.

3
Recombinant DNA( natural and manipulative)

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

4
Basics of 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.)

5
Process

• Restriction endonucleases cut at defined
  sequences 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. The physiological function of
  restriction endonucleases is to serve as part of
  system to protect bacteria from invasion by
  viruses or other organisms
• Cuts yield either "staggered" or "sticky" ends
  (see figure) or "blunt" ends.
• Two pieces of DNA cut with the same enzyme, can
  be pasted together using another enzyme called
  "DNA ligase".

6
Sticky ends

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

7

• Blunt ends
• (1) The restriction endonuclease cleaves in the
  center of the pseudopalindromic recognition site
  to generate blunt (or flush) ends.
•   HaeIII GG'CC
• HincII GTY'RAC

8
Restriction enzymes generate fragments that
facilitate recombination
9
Process

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

10
Restriction enzymes
11
Experimental Design
12
Recombinant DNA
13
Examples of Products of Genetic Engineering using
microbes

• Factor VIII
• Erythropoetin
• Insulin
• Interferon
• Epidermal growth factor

14
Industrial applications

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

15
Agricultural applications

• Frost resistant crops
• Insecticide resistant crops
• Herbicide resistant crops

16
Transformation with pGlo

• PRE-INCUBATIONThe recipent 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.
  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. 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.

17
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

18
pGlo

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

19
pGlo
20
pGlo
21
Gene fusion

• Transposition of genes from one location to
  another on a chromosome
• Also can result in the deletion of a section of a
  chromosome
• Gene fusion has been used with Pseudomonas
  syringae, a bacterium that grows on plants

22
Pseudomonas syringae

• Produce a protein that forms a nucleus for ice
  crystals
• Ice crystals damage leaves and stems
• Removing the gene, prevents damage to crops when
  the crops are sprayed with the resistant forms

23
Crop damage due to frost
24
P syringae on surface of leaves
25
Protoplast fusion

• Removal of the cell wall of organisms of two
  strains can result in the recombination of their
  genetic material.
• Can select for desirable features of both strains
• Effectively used in yeast, molds, and plants

26
Protoplast fusion

• Nocardia lactamdurans produces the antibiotic
  cephalomycin
• New strains increase the yield of this important
  antibiotic

27
Gene amplification

• Bacteriophages or plasmids are introduced into
  cells to repliicate or reproduce at rapid rates
• This is used particularly in strains of bacteria
  that produce antibiotics
• Amplification can also be used to increase the
  yield of amino acids, vitamins, and enzymes

28
Agrobacterium tumefaciens and natures genetic
engineering
29
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

30
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

31
Ti plasmid
32
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

33
Genetic Engineering and Ti