Microbio lab quiz 5

How did we test for the production of antibiotics by our isolates?

zone of inhibition

Alexander Fleming

Father of antibiotics for the accidental discovery of Penicillin

Molecular Clock

the average rate at which a species' genome accumulates mutations, used to measure their evolutionary divergence

What is thermal cycler

Used to amplify DNA sequence by increasing and decreasing temperature

Extension (PCR)

DNA polymerase synthesizes new DNA strands by extending the primers. 72C for 90s

Polymerase Buffer

creates optimum activity of Taqpolymerase

Tris-HCl

maintains pH

Triton

stabilizes DNA

Loading and Tracking buffer

bromophenol blue (stain), glycerol

coliphage

bacteriophage that infects E. coli

Paul Ehrlich

-magic bullet hypothesis

-developed concept of selective toxicity

-identified dyes that effectively treated African sleeping sickness

magic bullet hypothesis

Chemicals can be designed to bind to and kill specific microbes or tumor cells without harming the host itself

Selman Waksman

coined the term "antibiotic", developed the antibiotic streptomycin

Streptomycin

cure for tuberculosis

Alma Whiffen

discovered the antifungal agent cycloheximide

Carolus Linnaeus

Father of taxonomy, Domains of life

Phylogenetic tree

is the evolutionary relationship between taxonomic groups

Molecular phylogeny

Comparison of genetic sequences used to deduce relationships.

Cycloheximide

inhibits the growth of fungi

Antagonism and how did you detect it

antagonism- inhibition of action of one organism by another (one organism benefits while the other is harmed)

detection- zones of inhibition

How and why is antibiotic resistance a concern?

resistance to antibiotics makes it harder to treat severe infections

How does antibiotic resistance arise?

spontaneous mutations, horizontal gene transfer

Targets of antibiotics

cell wall, ribosomes, folic acid, cell membrane, DNA & RNA

ideal antibiotic

readily available, inexpensive, chemically stable, easily administered, nontoxic & nonallergic, selectively toxic against wide range of pathogens

broad spectrum antibiotics

affect a broad range of gram-positive or gram-negative bacteria

Narrow spectrum antibiotics

Effective against specific bacteria

PCR

polymerase chain reaction

16S rRNA

- small subunit of the ribosome of Bacteria and Archaea

- evolutionary information can be obtained;

-These are conserved genes

16S rRNA size

A large polynucleotide (~1500 bases)

What are the steps of PCR?

denaturation, annealing, extension

What is the purpose of PCR?

A laboratory technique for rapidly amplifying millions to billions of copies of a specific segment of DNA

Thermus aquaticus

is the organism that is thermophile whose enzymes are stable at high temperature

Taq polymerase

DNA polymerase which is heat stable taken from Thermus aquaticus, polymerization of dNTP into a DNA strand

dNTP's

mix of nucleotides building blocks of new DNAstrand

Denaturation

In proteins, a process in which a protein unravels and loses its native conformation, 95C for 30s

Anealing (PCR)

The temperature is decreased so that the primers can anneal to the complementary sequences on the DNA templates. 56C for 30s

What are primers?

small nucleotide sequence - locate target DNAfragments

Which primers were used for the 16S rRNA?

27F and 1492R

Buffer composition

Tris-HCl, EDTA, MgCl2, KCl, Triton

EDTA

chelating agent, keep DNA intact

MgCl2

magnesium chloride, cofactor for Taq polymerase

KCl

neutralizes charges in DNA template

PCR applications

Bacterial identification, DNA fingerprinting, bioengineering

What is the principle and purpose of Gel electrophoresis?

procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field, their charge, shape and size

DNA migration to opposite side from cathode to anode

DNA is negatively charged

What dictates how far a fragment will travel?

A porous agarose gel is used to slow the movement of DNA and separate by its size

What is agarose?

-first used in biology when Robert Koch*

-linear polymer extracted from seaweed

TBE buffer

(Tris/Borate/EDTA)

a liquid buffer that protects the DNA molecules and allows electricity to move through the chamber, driving the molecules across the gel

TAE buffer

tris-acetate w/ EDTA

good for DNA recovery

good for lg fragments

low buffering capacity

increases migration of DNA thru gel

Staining dye-Gel green stain

binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel

bromophenol blue (stain)

used to stain proteins and nucleic acids without UV

glycerol

loading buffer for weight

What is the DNA ladder?

a mixture of DNA fragments with known sizes.

How do we "read" the DNA migration?

Compare to known fragment sizes

plaques

zones of clearing from bacterial lysis, forming units (PFU)/ml

bacteriophage

A virus that infects bacteria

obligate intracellular parasites

cannot multiply unless they invade a specific host cell and instruct its genetic and metabolic machinery to make and release new viruses

titer

number of infectious units per volume of fluid

Coliphage and bacteria name used in lab

T4 and E.coli

lytic cycle

a viral reproductive cycle in which copies of a virus are made within a host cell, which then bursts open, releasing new viruses

lysogenic cycle

a viral reproductive cycle in which the viral DNA is added to the host cell's DNA and is copied along with the host cell's DNA

cloudy plaque

This means that there are some members of the bacterial population that are sensitive to this particular antibiotic, but others that are genetically immune to its effects. lysogenic

clear plaque

lytic

Calculation of titer

# of plaques x 1/dilution factor PFU/mL

PFU stands for what?

plaque foriming units

Starch test

Iodine solution turns blue-black

Which is the developer?

Iodine

Starch test negative

dark brown

Starch test positive

clearing zone