MBI201 Lab Final

Enterobacteriaceae inhabit

intestinal tract of mammals

Enterobacteriaceae characteristics

gram negative

non-spore forming

rod-shaped

some motile

Enterobacteriaceae examples

Citrobacter, Enterobacter, Escherichia, Salmonella, Serratia

metabolic capabilities

based on presence of absence of enzymes and pathways

carbohydrate fermentation

organic donor and final electron acceptor

bromcresol purple brother (BCP)

bromcresol purple as pH indicator

purple --> yellow under acidic conditions

BCP indicates

acidic waste from fermentation when color change from purple to yellow

Simmons citrate agar

Bromothymol blue as pH indicator

green --> blue under basic conditions

blue

positive Simmons citrate agar

purple

negative BCP

enterobacteriacae

mutliple-test system with 12 media that combines 15 biochemical tests useful in the identification of Enterobacteriaceae

advantage of enterotube

all tests are done simultaneously by inoculation from a single isolated coloony

metabolism

the study of what living organisms are chemically and how they use the raw materials in the environment to live, grow and reproduce.

biochemical fingerprint

the summation of the metabolic activities of an organism's enzymes

enzymes

detected by the chemical reaction they carry out

glucose medium

contained in an appropriate base medium, with cresol red as a pH indicator. The medium is covered with wax to provide anaerobic conditions and to allow detection of gas formation. Uninoculated: red.

lysine medium

contained in an appropriate base medium, with bromocresol purple as a pH indicator. The medium is covered with wax to provide anaerobic conditions. Uninoculated: yellow.

ornithine medium

contained in an appropriate base medium, with bromocresol purple as a pH indicator. The medium is covered with wax to provide anaerobic conditions. Uninoculated: yellow.

H2S/indole medium

Sodium thiosulfate (4.7 g/l), ferric ammonium citrate (0.6 g/l) , and tryptophan (1.2 g/l) in an appropriate base medium. Uninoculated: Beige to light amber.

adonitol medium

Adonitol (20.0 g/l) contained in an appropriate base medium, with cresol red as a pH indicator. Uninoculated: red.

lactose medium

Lactose (20.0 g/l) contained in an appropriate base medium, with cresol red as a pH indicator. Uninoculated: red.

arabinose medium

contained in an appropriate base medium, with cresol red as a pH indicator. Uninoculated: red.

sorbitol medium

contained in an appropriate base medium, with cresol red as a pH indicator. Uninoculated: red.

dulcitol/PA medium

contained in an appropriate base medium, with bromothymol blue as a pH indicator. Uninoculated: green.

urea medium

contained in an appropriate base medium, with phenol red as a pH indicator. Uninoculated: beige to light amber.

citrate medium

contained in an appropriate base medium, with bromothymol blue as a pH indicator. Uninoculated: green.

+ glucose fermentation

yellow (from red)

+ lysine and ornithine decarboxylation

violet (from yellow)

+ hydrogen sulfide production

black-brown (from beige)

+ adonitol, lactose, arabinose, sorbitol fermentation

yellow (from red)

+ dulcitol fermenation

yellow (from green)

+ phenylalanine deamination

dark brown (from green)

+ urea hydrolysis

purple (from beige)

+ citrate utilization

blue (from green)

plasmids

Small circular piece of DNA

One or many copies

inducible promoter

Only turned on when certain chemicals (inducer) are present in the media

advantages of plasmids in the lab

Introduce new genes

Select for cells by antibiotic resistance

Control gene expression

components of pGLO

arabinose, inducible promoter, GFP, bla (antibiotic resistance), origin of replication

GFP

green fluorescent protein

PCR

polymerase chain reaction

polymerase chain reaction (PCR)

a simple enzymatic assay which allows for the amplification of a specific DNA fragment from a complex pool of DNA.

advantage of PCR

It rapidly make millions to billions of copies (complete copies or partial copies) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it

steps of PCR

denaturation, annealing, extension

PCR components

DNA sample, primers, nucleotides, Taq polymerase, mix buffer, PCR tube

polymerase in PCR

taq

denaturing

strands separate

annealing

primers bind to template

extension

synthesis of new strand

Kovac's reagent

tryptophan degradation for indole test

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

how to analyze gel

UV light and coomassie stain

how to detect GFP in transformed cells

SDS-PAGE and analyzing gel

gel electrophoresis

separated charge particles (in protein) in an electric field due to pores in the gel

small proteins

run through gel more easily than

continuous polyacrylamide gel

stacking gel only

discontinuous polyacrylamide gel

stacking gel + resolving gel

stacking gel

upper portion, 4% acrylamide

resolving gel

lower portion, 5-20% acrylamide depending on your protein size

Laemmli buffer

Tris buffer, glycerol, SDS, 2-mercaptoethanol (BME)

glycerol

increases sample density

SDS

negatively charged detergent the binds protein and disrupts structure, equalizes charge density of proteins

charge density

charge/mass ratio

2 mercaptoethanol

BME or DTT, reduces disulfide bonds

SDS-PAGE result

linear/denatured proteins, equalized charge density (SDS) and proteins separated based on size/molecular weight

denaturation of GFP

reduces its fluorescent properties so you have to analyze partially denatured (no heat) and completely denatured (heated) protein preps

ladder

protein standards, mix of proteins with known molecular mass

expected sizes of proteins

37 and 27 kDa (which is completely denatured & which will fluoresce?)

fluorescence

GFP's chromophore absorbs light in 395 nm rand

how fluorescence works

chromophore electrons excited --> higher energy state --> as electrons drop back down to lower energy state they emit visible green light (509 nm range)

coomassie

stains all proteins

viruses

obligate intracellular parasites

Virus structure

nucleic acid coated with protein

envelope

a membranelike layer that covers the capsids of some viruses

capsid

protein coat that protects viral genomes

viral genome

DNA or RNA, single or double stranded

metabolic parasites

hijack the cellular machinery, uses energy, materials, enzymes, replication, transcription and translation

virion

a fully formed virus that is able to establish an infection in a host cell

lysis

destruction

viral budding

the release of virions from the host cell through the cell membrane, a process which includes encasing the virion with an envelope composed of lipids from the host cell membrane

icosahedral capsids

20 triangular sides

Each triangle made up of at least 3 identical capsid proteins

Allows small protein to cover a large volume

(Herpes)

filamentous capsids

Long tube of protein, with genome inside (Ebola)

complex capsids

Mixture of icosahedral and filamentous shapes

Many bacteriophages

asymmetrical (irregular) viral shapes

tend to be larger viruses (poxvirus)

group 1

dsDNA

group 2

ssDNA

group 3

dsRNA

group 4

+ ssRNA

group 5

- ssRNA