Transcript Recombinant Technology

Recombinant DNA,
Biotechnology, and Microbes
Microbiology 221
Overview – Putting microbes to
Work – Molecular Cloning
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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.
Recombinant DNA( natural and
manipulative)
Combination of DNA from organisms
from two different sources
 Bacterial and human
 Bacterial and plant
 Viral and human
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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.)
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Process
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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".
Sticky ends
Sticky ends
 When the ends of the restriction
fragments are complementary,
EcoRI – recognition sequence
5'---G ‘AATTC---3'
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3'---CTTAA ‘G---5'
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Blunt ends
(1) The restriction endonuclease
cleaves in the center of the
pseudopalindromic recognition site to
generate blunt (or flush) ends.
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HaeIII
GG'CC
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HincII
GTY'RAC
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Restriction enzymes generate fragments that
facilitate recombination
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
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Restriction enzymes
Experimental Design
Recombinant DNA
Examples of Products of Genetic
Engineering using microbes
Factor VIII
 Erythropoetin
 Insulin
 Interferon
 Epidermal growth factor
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Industrial applications
Oil “ eating” microbes – Prince William
Sound – Alaska
 Degradation of mercury in the
environment – Clean up of
contaminated sites
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Agricultural applications
Frost resistant crops
 Insecticide resistant crops
 Herbicide resistant crops
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Transformation with pGlo
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PRE-INCUBATION
The 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.
* 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.
* 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.
Plasmid Vectors
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Ori( origin of
replication)
Polylinker cloning sites
Regulatory region
( lac operon)
Antibiotic resistance
gene(s)
Reporter gene for
protein – color or
fluorescent molecule
pGlo
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Ori
Polylinker cloning
region
Amp ( beta lactamase
for resistance)
araC( arabinose operon)
pBad
Green fluorescent
protein - reporter
pGlo
pGlo
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
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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
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Crop damage due to frost
P syringae on surface of leaves
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
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Protoplast fusion
Nocardia lactamdurans – produces the
antibiotic cephalomycin
 New strains increase the yield of this
important antibiotic
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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
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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
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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
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