Microbiology: Chapter 9

tanktherapistBiotechnology

Oct 23, 2013 (4 years and 21 days ago)

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Microbiology: Chapter 9

Biotechnology and

Recombinant DNA



Biotechnology
: The use of
microorganisms, cells, or cell
components to make a
product.


Foods, antibiotics, vitamins,
enzymes


Recombinant DNA (
rDNA
)
technology
: Insertion or
modification of genes to
produce desired proteins

E. coli

Biotechnology and Recombinant DNA


Vector
:
Self
-
replicating DNA
used to carry the
desired gene to a new cell


A bacterial plasmid or viral genome is used


Clone
:
Population of cells
arising from one
cell, each
carries the new gene or plasmid


A Typical Genetic Modification
Procedure

A Typical Genetic Modification
Procedure

Selection and Mutation


Selection
: Culture a naturally occurring
microbe that produces desired product


Mutation
: Mutagens cause mutations that
might result in a microbe with a desirable trait


Site
-
directed mutagenesis
: Change a specific
DNA code to change a protein


Select and culture microbe with the desired
mutation

Restriction Enzymes


Cut specific sequences of DNA


Fragments of DNA produced by the same
restriction enzyme will spontaneously join by base
pairing



DNA ligase can covalently link the DNA
backbones.


Destroy bacteriophage DNA in bacterial cells


Cannot digest (host) DNA with methylated
cytosines

ANIMATION: Recombinant DNA Technology

Restriction Enzyme & Recombinant
DNA

Vectors


Carry new DNA to desired cell


Shuttle vectors

can exist in several different
species


Plasmids and viruses can be used as vectors

A Plasmid Vector Used for Cloning

Polymerase Chain Reaction (PCR)


To make multiple copies of a piece of DNA
enzymatically


Used to


Clone DNA for recombination


Amplify DNA to detectable levels


Sequence DNA


Diagnose genetic disease


Detect pathogens

ANIMATION PCR: Components

ANIMATION PCR: Overview


Can identify an
unknown
bacterium
because the RNA
primer is specific

PCR

PCR

PCR

ANIMATION PCR: Process

Inserting Foreign DNA into Cells


DNA can be inserted into a cell by


Electroporation :
a significant increase in the
electrical conductivity and permeability of the cell
plasma membrane caused by an externally applied
electrical field.


Transformation:
the transfer of genetic information
from dead bacteria to live ones


Protoplast fusion
: type of genetic modification in
plants by which two distinct species of plants are
fused together to form a new hybrid plant with the
characteristics of both.

Process of Protoplast Fusion


Valuable in the genetic
manipulation of plant
& algal cells

Inserting Foreign DNA into Cells


DNA can be inserted into a
cell by


Gene gun :
a device for
injecting cells with genetic
information.


Microinjection:

process of
using a glass micropipette to
insert substances at a
microscopic into a single living
cell.

Microinjection of Foreign DNA

Obtaining DNA


Genomic
libraries

are
made of pieces
of an entire
genome stored
in plasmids,
phages, or yeast
DNA


Obtaining DNA


Complementary
DNA (cDNA)

is made
from mRNA by
reverse
transcriptase


cDNA is important
because
it lacks
introns

Obtaining DNA


Synthetic
DNA

is
made by a
DNA
synthesis
machine

Selecting a Clone

Selecting a Clone

Selecting a Clone

Selecting a Clone

DNA Probe


A single
-
stranded DNA molecule used in
laboratory experiments to detect the presence
of a complementary sequence among a
mixture of other singled
-
stranded DNA
molecules


Used to identify bacteria carrying a specific
gene

Making a Product

E. coli


Used because it is
easily grown
and its
genomics are
known


It’s
endotoxin

eliminate from
products


Cells must be
lysed

to get product

Making a Product

Saccharomyces cerevisiae


Used because it
is easily
grown and its genomics
are known


May express eukaryotic
genes easily





Mammalian cells


May express
eukaryotic genes
easily


Harder to grow



Plant cells and whole plants


May express eukaryotic genes easily


Plants easily grown

Therapeutic Applications


Human enzymes

and other proteins (such as
human insulin)


Subunit vaccines


Nonpathogenic viruses carrying genes for
pathogen's antigens as
DNA vaccines


Gene therapy

to replace defective or missing
genes

The Human Genome Project


Nucleotides have been sequenced in human
DNA


Human Proteome Project

may provide
diagnostics and treatments


Reverse genetics
: Block a gene to determine its
function

Scientific Applications


Understanding
DNA


Sequencing
organisms'
genomes


DNA
fingerprinting
for
identification
(source of viral
or bacterial
pathogens)

Southern Blotting

Southern blotting can be used to locate a
gene in a cell.

Southern Blotting

Southern Blotting

Forensic Microbiology


PCR:
polymerase chain reaction

-

is a
technique to amplify a single or few copies of
a piece of DNA across several orders of
magnitude, generating thousands to millions
of copies of a particular DNA sequence.


Primer for a specific organism will cause
application if that organism is present

Forensic Microbiology


Real
-
time PCR
: Newly made DNA tagged with
a fluorescent dye; the levels of fluorescence
can be measured after every PCR cycle


Reverse
-
transcription (RT
-
PCR)
: Reverse
transcriptase makes DNA from viral RNA or
mRNA



Nanotechnology


Study of the
controlling of matter
on an atomic and
molecular scale


Bacteria can make
molecule
-
sized
particles


Using
Agrobacterium


Uses horizontal
gene transfer to
cause tumors in
plants


Herbicide resistance


Suppression of
genes


Antisense DNA


Nutrition


Human proteins

Using
Agrobacterium

Gene Therapy


The correction of a genetic deficiency in a cell
by the addition of new DNA and its insertion
into cells or tissues to prevent disease


In the future, it is hoped that by inserting the
insulin gene in a diabetic person’s pancreatic
cells, Type I diabetes can be cured


Safety Issues and Ethics of Using rDNA


Avoid accidental release


Genetically modified crops must be safe for
consumption and for the environment


Who will have access to an individual's genetic
information?