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46 Cards in this Set

  • Front
  • Back
Group A Streptococcus

- Synonymous with the species Streptococcus pyogenes (Toxins for Scarlet Fever - M Protein) - are important in common human pathogens

Streptococcal pharyngitis
Also known as strep throat
Impetigo

Surface skin infection

Streptococcal necrotizing fasciitis
Flesh-eating strep

- Strep throat


- Scarlet fever


- Flesh-eating strep


- Impetigo

4 Diseases Group A Streptococci can cause

- Rheumatic Fever


- Glomerulonecrophitis (Immunocomplex hypersensitivity)

- When cross-reacting antibodies to Group A streptococcal proteins, it can lead to autoimmune conditions such as (2)
Strep Alert
- Rapid test for the detection of Group A antigen on the surface of Streptococcus pyogenes

Rapid Strep Tests

- Rapid Antigen Detection Test (RADT) that cannot distinguish between GAS infections and asymptomatic carriage


- Throat culture should be used to confirm GAS infection, but antibiotics start immediately after results come out in 24-48 hours

Negative


Positive


Invalid

Testing for Group A antigens


- One pink band in "Control Band" Area near handle means?


- Two pink bands; "Control Band" and "Specimen Band"


- No pink "Control Band"

- Extract DNA from sample of bacterial cells isolated from patients and potential sources of infection


- Polymerase Chain Reactions to duplicate and make copes for region of the gene which codes for a protein in the bacterial flagellum


- Digest half of PCR product with restriction enzyme


- Stain and separate the fragments of DNA with digested and undigested samples using agarose gel electrophoresis to create visual DNA fingerprint of bacterial samples


- Compare fingerprints


5 Steps of the PCR-RFLP Experiment

- Determine the source of an outbreak of food-borne E. coli gastroenteritis
What was the purpose of PCR-RFLP?

Clear Liquid


White Pellet

After the bacteria strain is mixed with an alkaline DNA extraction solution and boiled to lyse the cells, then centrifuged, what part of the centrifuge is the DNA in? The cell debris? (Protein, carbohydrates, RNA)

- So DNA Polymerase will recognize where to attach; and to design specific primer even if there are different strains


- Variable to keep changing to avoid immune systems; to analyze different strains

Why does gene have stable/constant and variable regions?
If it's too short there is no variation and the electrophoresed bands are too similar
flic gene was about 1,500 base pairs. What would happen if it were much shorter?
fliC gene

- Controls the production of the protein flagellin


- One of the major antigens present in gram negative bacteria


- Responsible for physical structure and helical nature

- Bacteria


- To attack bacteriophages and their viral DNA





Restriction Enzymes come from where? To usually do what? (Besides digest DNA)



Taq polymerase

The thermophile Thermus aquaticus provided us with a unique DNA Polymerase that is specific, and heat-stable


- This is called?

- Taq polymerase


- Mg+2 ions


- Mixture of nucleotides


- Buffer


- DNA template


- Set of RNA primers for a specific gene (Forward and reverse)

The following 6 things must be present for the polymerase to copy a DNA template
Magnesium ions
- Act as cofactors which allow the polymerase to function properly
RNA primers

- short 20-30 pieces of DNA that bracket each end of the gene


- Designed to be specific to the gene being studied

Thermal Cycler

- Cycles the samples through a series of different temperatures


* 95C for 1 minute to melt DNA and separate strands


* 55-60C for 30-60 seconds so forward/reverse primers can bind to each strand of DNA


* 65C to allow the Taq polymerase synthesize DNA (1 minute per 1000 bases so time can vary)




Repeat 30-40 times for 2-3 hours to create billions of bracketed genes

RsaI


5' GT- AC 3'


3' CA - TG 5'



The restriction enzyme that is used for the experiment that was isolated from bacteria Rhodopseudomonas sphaeroides?


Where does it cut?

- Stabilizes the pH of the DNA molecules (Slightly negative and water soluble)


- Controls the amount of electrical current passing through chamber

Purpose of buffer in electrophoresis chamber?

Weighting Agent


Ficoll



- Added to increase the density of the sample which will cause it to sink into the well instead of dispersing out into the buffer


- Name of the one we used?

Colored loading dye


dye cresol red


Sybr-Gold

- Added to make the sample easier to see when loading into the gel, doesn't actually stain the DNA


- The one we used?


- The stain we used?

- Adds a little weight to sink into well


- Stain the DNA molecules


- Causes them to fluoresce when exposed to UV light

3 Reasons for using the Stain Sybr-Gold?

125V


20 Minutes


Three-Fourths

What is the voltage and time we set the electrophoresis for?


If the pink bands reach ________ of the chamber, turn off the chamber.

"Ladder" - Gene Ruler

- Composed of a known mixture of DNA fragments ranging from 100-3000 base pairs in size


- Gives standard by which to judge size of the DNA fragments in the samples of the gel

Streptococcus pneumoniae


Smooth-encapsulated


Rough-no capsule

Frederick Griffith's experiment with mice involved what bacteria?


Which were pathogenic? Which were not?

Avery, McCarty and Macleod
- 3 Scientists who demonstrated the "transforming principle" was DNA from the nonliving smooth cells being incorporated into the living rough cells in Griffith's experiment

Transformation
Defined as the incorporation of naked, cell-free DNA into a competent host cell
Competency

- Defined as the ability of a cell to receive extra-cellular molecules of DNA

- Prevent incorporation of foreign DNA into the host genome


- Prevent parasitism from bacteriophages

2 Functions of restriction enzymes that destroy DNA that they recognize as foreign, such as viral bacteriophage DNA

Neisseria gonorrhea (Opa protein)


Haemophilus influenza (Capsule)

2 significant pathogenic bacteria have surface proteins that bind species similar DNA in the environment and transport inside of the cell


In particular, able to avoid host immune response by varying pili structure so it can keep reinfecting

Transformation


Recombination

- 2 mechanisms of bacteria that accomplished the same introduction of species variance as sexual reproduction does in higher species

1. Change ionic strength of medium with Ca2+


2. Heat shock by rapid temperature fluctuation


3. Electroporation- high voltage to render cells permeable to DNA

3 Artificial Conditions to make bacteria more competent without the use of DNA binding proteins

pGreen


Green Fluorescent Protein

- Plasmid that we will be using __________ that contains the gene ______________ from a bioluminescent jellyfish Aequorea Victoria to act as a reporter molecule


- This plasmid also contains gene for beta-lactamase

Ampicillin resistance (Antibiotic)
Beta-lactamase gene in the pGreen plasmid will also distinguish it by giving the bacterial cell ________________
E. coli

- Montezuma's Revenge


- Fatal hemolytic uremic syndrome


There are examples of infections that can be caused by genes from different strains of ____________

Selective Media

- Use of this media to distinguish if there was transformation or not


- Results can also indicate if there may have been spontaneous mutation


FAILSAFES

- Don't grow without host cells
- Why is it difficult to culture viruses?

T4


phi-X174

- Bacteriophages that we work in which one has


- Tail that is important in host recognition and inject of viral genetic material


- One that has bacterial pilus that it follows to host cell and then penetrates

Lytic Reproduction Cycle

1. Genetic material of phage directs host cell to destroy its own DNA and manufacture/assemble new phage particles


2. Cell will rupture and release new phage particles




- About 30 minutes at 37C



Plaques
- Clear zones within bacterial lawns that are the only visual indication of presence of viruses - by the death they cause
Neutralize
- Antibody binding to the part of the virus that attaches to the host cell, rendering virus noninfective
anti-TF antibody serum

This serum is made by


- Injecting T4 phage into animal over several months, cannot infect animal cells, but antibodies will result