Microbiology & Microbiology & DNA TechnologyDNA Technology
Mrs. DanielsAdvanced Biology - Ch. 23 &
14Modified April 2008
MicrobiologyMicrobiology
• 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 .VIRUSES .
• 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
VIRUSESVIRUSES
• 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
VIRUSESVIRUSES
• NOTE the list of animal infecting types of viruses in your book.
• 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 BACTERIOPHAGES . .
• Sometimes just called phages
• Capsid
• Tail
• Tail fibers
• Mode of infection: insert their nucleic acid (typically ds DNA)
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
Lysogenic CycleLysogenic 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 CycleLysogenic 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
PrionsPrions
• 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
ProkaryotesProkaryotes• 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
ProkaryotesProkaryotes• 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 (disease-causing 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
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 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
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.