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)

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

TheLysogenic

Cycle

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

Cocci

Bacilli

Spirilli

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

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 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.