Download Microbiology

Microbiology &
DNA Technology
Mrs. Daniels
Advanced Biology - Ch. 23 & 14
Modified April 2008
Microbiology
• Typically, microbiology encompasses all
life-like and living organisms that do not fit
into the categories of the macroscopic
“large-scale” world
• Includes prokaryotes (eubacteria and
archaebacteria), viruses, prions, and
protists
• Also some unicellular fungi (yeast), plants
(algae), and animals (daphnia)
VIRUSES
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What is a virus?
Is it alive?
Parts:
Capsid - protein coat
– Helical or a polyhedron (or a combination)
• Outer membranous envelope (on some)
• Nucleic acid - either DNA or RNA
– May be ss or ds
VIRUSES
• Requires a host to reproduce
• Where did they come from?
• Some scientists believe that viruses originated
as “part” of certain cells and that they must
have “escaped” from those cells
• This explains the specificity that a virus has to
its host
VIRUSES
• NOTE the list of animal infecting types of viruses in
your book.
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Bacteriophages:
Viruses that attack bacteria
Important in genetic research
Can be used clinically to kill pathogenic bacteria
Reminder: Pathogens are any agents that cause
disease
BACTERIOPHAGES
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Sometimes just called phages
Capsid
Tail
Tail fibers
Mode of infection: insert their
nucleic acid (typically ds DNA)
In the time it
takes to eat
lunch…
The LYTIC cycle
• 1. Attachment - phage attaches to bacterial
cell wall
• 2. Penetration or Entry - the DNA is
inserted into the bacterium
• 3. Replication - the virus parts are copied
• 4. Assembly - the virus parts are put
together to make new bacteriophages
• 5. Release - release of the new phages
The
Lysogenic
Cycle
Lysogenic Cycle
• Some bacteriophages don’t immediately lyse their
host cell
• They can insert their DNA into the host’s DNA.
• Now called a prophage
• When the lysogenic cell (host cell) begins to exhibit
the characteristics of the viral DNA (seen as new or
unusual properties) then “conversion” has occurred.
Lysogenic Cycle
• How does this affect us?
• The bacterium that cause certain diseases sometimes
only cause them when they themselves have been
infected by a virus (bacteriophage)
• Ex. Diphtheria
• Ex. Botulism
Viroids
• Are these alive?
• Smaller, YES SMALLER than viruses!
• Contain no proteins nor genes to code for
proteins
Prions
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Are these alive?
Prions are also smaller than viruses
Proteinaceous infectious particles
Made of protein and NO nucleic acid
Responsible for transmissible spongiform
encephalopathies
• Mad cow disease (and Creutzfeldt-Jakob)
• Chronic wasting disease
Prokaryotes
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Include Archaea and Eubacteria
Most are unicellular, but some form colonies or filaments
Are these alive?
Small cells
Shapes: coccus, bacillus, spirillus, or shapeless
Cell walls - to help them thrive in hypotonic media
Eubacteria:
If they have thick peptidoglycan walls, they stain purple
(Gram +)
• Thin peptidoglycan walls but thick outer layer of lipids and
carbohydrates, they lose the purple and retain pink (Gram -)
• Some have capsule, endospore, or pilus
Prokaryotes
Bacilli
Cocci
Spirilli
Prokaryotes
• If they lack peptidoglycan completely in their cell
walls, they are probably Archaea
• Single circular, highly folded DNA molecule
• Plasmids
• Binary fission
• Heterotrophs: saprobes or symbiotic (diseasecausing parasites)
• Autotrophs: chemo- or photo-
• Most proks are aerobic
• Some are facultative anaerobes and some are
obligate anaerobes
• Exchange of DNA (not sexual reproduction) there are three ways
• Transformation - fragments taken up from a
damaged cell or from the environment
• Transduction - transferred by bacteriophage
• Conjugation - exchange through pilus
DNA Technology
Bacteriophages
 Bacteriophages laid the foundation for
recombinant DNA methods
 Restriction enzymes- molecular scissors
 Recognition sites - palindromic sequences
 AAGCTT and its complement TTCGAA
 “sticky ends”
 Joined with ligases
Vectors
 Recombinant DNA is formed when DNA is
spliced into a vector
 Common vectors: bacteriophages, plasmids, of
BAC’s (bacterial artificial chromosomes)
 Temporarily houses the DNA
Transformation
 The process of making the bacterial cell wall
permeable to the plasmid is called transformation
 Puncturing the cell wall
 Chemically altering
 Heat shock
 We’ll be conducting a bacterial transformation lab
where we splice two genes into E.coli
Libraries
 How can you locate the gene of interest that you
want to splice?
 Genomic library - fragments of all the DNA in a
genome
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Put one of each human gene into a bacterial
plasmid
 Chromosome library - all DNA fragments isolated
from individual chromosomes
Easier to use this to find your gene
 DNA fingerprinting & human genome project helped
us to locate many genes on each chromosome
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Gene Splicing
 Choose the gene you’d like to splice and locate
it
 Cut it out with restriction enzymes
 Cut the vector using the same restriction
enzymes
 Mix the two types of DNA (ligase joins them)
 Transform the bacterium
 Allow the bacterium to reproduce
 Test to determine effectiveness
Electrophoresis
 The other half of AP Lab 6 deals with DNA
fingerprinting or electrophoresis
 Agarose - seaweed
 Filters DNA according to fragment length
(molecular weight)
 Filters other molecules according to molecular
weight, (size and shape), and charge
 Use stain to see results OR use radioactive
DNA probe and UV light
Electrophoresis
 DNA with radioactive probe - Southern blot
 RNA - Northern blot
 Protein or polypeptide molecules - Western blot
 One well-known use is to detect antibodies, such
as antibodies to HIV
 Getting enough DNA to run in an electrophoresis
requires amplification
 Make a lot more of the DNA samples
PCR
 Polymerase Chain Reaction
 Heat the DNA - separate the strands
 Cool
 Add DNA polymerase (from Thermus
aquaticus) and primers
 repeat
Sequencing
 Another important purpose of electrophoresis is
sequencing
 Chain Termination Method:
 Radioactively labeled primers
 DNA polymerase
 One of each of the four dideoxynucleotides
 These stop the addition of nucleotides to the
chain; therefore, they cause the chain to stop
when they are incorporated into a new strand
 This fragment (and all of the others) can then be
separated based on fragment length
Sequencing
 Automated:
 Much is now done by computers and machines
using fluorescent dyes instead of radioactive labels
 ~1.5 million bases decoded in 24 hours
 Human genome = 3 billion base pairs
 Genomes of over 100 organisms have been
sequenced (as of 2003)
 RFLPs (restriction fragment length polymorphisms)
- comparison and measure of genetic relationships
between genomes of different organisms
Applications of Genetic
Engineering
 Identifying genetic mutations
 Gene therapy
 Engineered proteins (ex. Insulin, growth hormone,
clotting factor VIII, etc.)
 Transgenic animals and engineered proteins
 Determining the role of a particular gene
 Agricultural
 Environmental
 Criminal justice
 Research, cloning, medicine, etc.