MCB 3020L CH 20-23

One method is the treatment of bacterial cells with an ice-cold calcium chloride (CaCl₂) solution

which enables the cells to uptake DNA from their environment; The drastic treatment with CaCl₂, allowing DNA to pass through the membrane and into the cell, alters its permeability.

Electroporation

Another membrane altering method where cells are subjected to high voltage electric impulses that destabilize the cell membrane, resulting in increased permeability and enabling DNA to pass into the cells

Plasmid

small, circular piece of extrachromosomal, double-stranded DNA capable of independent replication

Plasmids replicate

independently of the bacterial chromosome and range in size from a few thousand base pairs (bp) to more than 100 kilo base pairs (kb)

Plasmid Cloning Vectors

are plasmids that are used for transformation experiments and, therefore, have been genetically engineered. For example they contain an antibiotic resistance gene and a region in which exogenous DNA fragments can be inserted.
Vary in size

pGLO Plasmid

The plasmid that is used for the transformation of E . coli, which contains several reporter genes

ori

origin of replication

Green Fluorescent Protein (GFP)

generates bioluminescence under UV light

AraC gene

regulates the expression of GFP in the presence of the sugar arabinose

Beta-lactamase antibiotic resistance (bla) gene

allows for selection of positive transformants by plating on ampicillin plates

The real-life source of the GFP gene is

the bioluminescent jellyfish

Transformed cells will appear

white (wild-type phenotype) on plates not containing arabinose and fluorescent green under UV light when arabinose is included in the nutrient agar

Viruses

are obligate intracellular parasites that are incapable of reproduction outside of a host organism.

 Even the most complicated viruses have a relatively simple structure consisting of a protein coat

containing nucleic acids.

Viruses genetic information can be

dsDNA, ssDNA, ssRNA, or dsRNA

Viruses don’t need to maintain a vast amount of genetic information because

they reproduce by using the host cell’s synthetic machinery and materials

Lytic pathway

initiated by virulent viruses, the viral nucleic acids direct the cellular metabolism to produce new viral proteins and new copies of the viral nucleic acids.  These are assembled within the host cell until the cell is lysed and the new virus particles are released.

Lysogenic pathway

Temperate viruses incorporate their genetic material into the host cell’s genome. When the host cell reproduces it also reproduces the viral nucleic acids and the daughter cells contain the copies of the viral genetic material.

A lytic infection

leaves a dead host cell and up to several hundred new infectious virus particles available to infect surrounding cells and repeat the process

Lysogenic cycle stays the same until

conditions are favorable for the virus to be released at which point the viral nucleic acids will direct the cellular metabolism to produce new virus particles and switch to the lytic cycle

Steps for viral replication

attachment, penetration, protein synthesis, assembly, and release

 Each virus relies on a specific host.  Some viruses are specific to bacterial hosts, so they are classified as

bacteriophage or just the shortened term “phage.”

Capsid

contains the nucleic material

 Proteins on the tail fibers

are specific for receptors on the surface of a bacterium

The virus particle attaches to the cell surface using those receptors and

the tail sheath contracts to perforate the cell wall with the viral pin.

Provide lawn of bacteria

Suitable for virus

Plaque

Clear zone of lysed bacterial cells; One plaque = one viral particle

Titer

Concentration of virus particles

Molten top agar- ECB and virus added; Must not be kept over 50°C- why?

Temperatures significantly above 50°C can cause irreversible damage to the bacterial cell membrane and denature essential proteins/enzymes, killing the host cells.

Each individual has a unique DNA profile, though

humans are 99% identical

For every 1000 nucleotides inherited

there is 1 site of variation or polymorphism

Polymorphism

site of variation in DNA; Single-nucleotide polymorphism (SNP) or involve thousands of nucleotides

Restriction Enzymes

produced by bacteria as a defense mechanism against phage infection

Restriction enzymes recognize 4 - 8 bp sequences called

Restriction Sites and cleave DNA at these sites

Since restriction enzymes cleave DNA within the molecule

they are also called DNA endonucleases

Restriction Fragments

DNA cut by restriction enzymes, reproducible set of fragments

The process of producing restriction fragments

Each restriction enzyme/endonuclease is named for the genus (first letter) and species (second and third letter) of the organism it was isolated from. There are hundreds of different restriction enzymes that recognize and cut their specific sequences in a DNA molecule. Once the cuts have been made, the DNA molecule will break into a reproducible set of fragments

RFLPs

result from restriction enzymes cutting DNA

Loss of a site

appearance of larger fragment

Formation of a site

loss of larger fragment; two new smaller fragments

DNA fingerprinting uses?

Convicting felons and establishing familial relations

Human genomic DNA

around 3 billion bp

Why is the length of human genomic DNA a problem for fingerprinting?

Too long and restriction pattern can’t be seen (usually a smear)

Bacterial DNA

over 4 million bp

Lambda (λ) phage DNA

Linear, approximately 50 kbp, Clear bands on gel

Number of fragments after restriction:

Linear DNA= # of sites + 1, Circular DNA= # of sites

How many DNA fragments would you expect?

A circular piece of DNA w/ 3 cut sites; A linear piece of DNA w/ 3 cut sites

Agarose Gel Electrophoresis

used to separate fragments based on size

DNA phosphate groups

carry negative charge

• Cumulative with longer molecule having greater “-” charge

Agarose gel electrophoresis requires

Power supply, Agarose gel, TAE buffer, Ethidium bromide

Power supply

Electricity travels from cathode (-) to anode (+)

DNA is loaded near cathode- why?

DNA is negatively charged (due to its phosphate backbone).

When the electric current is applied, the DNA is repelled by the negative cathode and attracted toward the positive anode, forcing it to migrate across the gel.

Agarose gel

Agarose- component of agar; no charge

TAE buffer

conducts charge

Ethidium bromide

visualize DNA

What causes the movement of fragments

Electricity

Smaller fragments travel

easier and faster

Higher agarose %

more restrictive, separates small fragments of DNA more easily

Lower agarose %

separates larger fragments of DNA more easily

Tracking/loading dye/sample buffer:

charged and will show when to stop applying current

Tracking/loading dye/sample buffer contains

bromophenol blue (tracking dye) and glycerol (increases sample density)

Fragments can run off gel

Same size fragments

travel together and form bands

Ethidium Bromide (EtBr) is needed to

visualize bands

Binds to DNA and fluoresces (UV transilluminator)

Carcinogenic

Staphylococcus aureus & Staphylococcus epidermidis

Gram-positive genus

Staphylococcus aureus can

can ferment mannitol and cause beta-hemolysis on blood agar

Staphylococcus aureus also

Produces an extracellular thermostable nuclease encoded by nuc gene

Mannitol salt agar (MSA)

Selective and differential medium

MSA contains

peptones, beef extract, agar, mannitol, phenol red, and 7.5% NaCl

MSA phenol red pH indicator

Yellow below pH 6.8, Pink above pH 7.4

What is MSA selective and differential for?

Selective for salt tolerant bacteria (Mainly Staphylococci), Differential for mannitol fermenting bacteria( i.e. Staphylococcus aureus)

If a species is able to ferment mannitol, what color will the medium turn? Why?

Yellow, because when a microorganism ferments the carbohydrate mannitol, it produces acidic metabolic byproducts.

Blood agar

Can be used as enriched or differential medium

○ In this lab- differential medium

5% sheep blood

Allows analysis of hemolytic capabilities

Hemolysins (exoenzymes)

lyses red blood cells and degrade hemoglobin

Three major types of hemolysis

○ Beta (β)- beta-hemolysin, completely breaks down RBC and

hemoglobin, clear zone around colony

○ Alpha (α)- alpha-hemolysin, partially breaks down RBC,

greenish halo around colony

○ Gamma (γ)- not able to lyse RBC, no change in medium