IBI Exam 1

Ames test

• developed in the 1970’s by Bruce Ames, Professor of Biochemistry at UC-Berkeley

• fast, sensitive, easy, and inexpensive (it doesn’t use a live animal model) assay of the ability of a chemical compound or mixture to induce mutations in DNA

• Measures the ability of a chemical to induce back mutations, or reversions, of an auxotroph to its original prototrophic state.

phototrophs

Bacteria that are able to synthesize all of the biochemicals needed for their own growth. They contain hundreds of metabolic pathways, each working to synthesize a single biochemical molecule.

The “wild type”, capable ofsynthesizing its own nutrients and growing on minimal medium

strain

a genetic variant or subtype of a microorganism

auxotroph

a particular strain or sample of bacteria unable to synthesize a needed molecule. In order for the bacteria to grow, the missing molecule must be added to the media.

The “mutant”, incapable of producing essential nutrients, and can only grow in complete media

reversions

“back mutations” of an auxotroph to its original phototrophic state

The greater the number of revertant colonies, the ___ mutagenic the test substance must be.

more

histidine

an amino acid needed for protein synthesis

limitations of the Ames test

• the rate of mutagenicity isn’t the same in all organisms due to differences in the rate of chemical absorption by the cells and different metabolism of compounds

• in some cases, the compound isn’t mutagenic but the metabolites of the compound are

• compounds testing positive in this version of the Ames test would be subject to testing in mammalian model systems

metabolite

a substance formed during the metabolism of a compound

s. typhimurium strain TA1535

• T to C missense mutation in the hisG gene, leading to a leucine to proline amino acid substitution

• defect in the lipopolysaccharide cell wall causing them to be more vulnerable to exogenous mutagens

• defective DNA excision-repair mechanisms, which would normally correct mutations arising during DNA replication or from exogenous mutagens

s. typhimurium strain TA1538

• deletion of one base pair (c) in the hisD gene, causing a 1 frameshift mutation and bringing a stop codon into the reading frame prematurely

• defect in the lipopolysaccharide cell wall causing them to be more vulnerable to exogenous mutagens

• defective DNA excision-repair mechanisms, which would normally correct mutations arising during DNA replication or from exogenous mutagens

To quantitatively determine the mutagenicity of a compound, one must determine:

• the number of mutant bacteria produced

• the total number of cells exposed to the mutagen to begin with

binary fission

the process by which our bacteria doubled for about 10 hours

bacterial colony

consists of multiple microorganisms that are all from one mother cell which all gather together and are genetically identical

formula for CFU/mL

number of colonies * reciprocal of dilution factor / volume plated in mL

Serial dilutions: mL test sample, mL broth, dilution, dilution factor, reciprocal of dilution factor, amount plated (mL)

1.0 mL test sample, 9.0 mL broth

Dilution: 1:10, 1:100, 1:1,000, 1:10,000, 1:100,000, 1:1,000,000

Dilution factor: 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6,

Reciprocal of dilution factor: 10¹, 10², 10³, 10⁴, 10⁵, 10⁶

Amount plated: 1 mL

lawn

uncontrolled bacterial growth, which happens when we don’t reduce the concentration of a sample before plating it 

culture media

Contains all the elements that most bacteria need for growth. Used for general cultivation and maintenance of bacteria.

Ex: Nutrient agar, LB broth, Trypticase soy agar

minimal media

A defined media that has just enough ingredients to support growth. Used to select for or against certain microorganisms

selective media

Contains or lacks a specific component that is needed to survive in that media. Used to separate specific microorganisms from others

ex: LB with ampicillin, HIS- media 

differential or indicator media

Distinguishes one microorganism from another growing on the same medium. Uses biochemical characteristics microorganisms as indicators of their presence.

ex: Blood agar, Eosin Methylene blue, Mannitol salt agar

genotoxicity

the ability of a chemical to damage genetic information(which may lead to mutations and/or cancer)

If the reversion frequency of the tested product is at least 5 times higher than that of the Negative Control, then the tested product is genotoxic. 

silent mutation

TTC → TTT

a point mutation that changes a codon but not the protein it produces

missense mutation

TTC → TCC

a point mutation that codes for a different protein

nonsense mutation

TTC → ATC

a point mutation that prematurely introduces a stop codon

insertion

a frameshift mutation in which a codon is added

deletion

a frameshift mutation in which a codon is removed

lysis

releasing of DNA from the cell

steps of DNA purification

1. DNA lysis

2. Purification of DNA from contaminants

*Successful DNA isolation requires methods that prevent nuclease degradation of the DNA

InstaGene Matrix

• a commercially available kit to isolate genomic DNA, made with a specially formulated 6% w/v Chelex resin

• bonds to PCR inhibitors, preventing DNA loss due to irreversible DNA binding

• absorbs cell lysis products that interfere with the PCR amplification process

Polymerase Chain Reaction (PCR)

• invented in 1938 by Kary Mullis, a scientist who worked for a biotech company based in the San Francisco area

• valuable in DNA fingerprinting, detection of bacteria or viruses (particularly AIDS), and diagnosis of genetic disorders

What’s needed for PCR? 

template: the DNA you want to amplify

Primers: short pieces of single-stranded DNA that are complementary to the target sequence

Nucleotides: (dNTPs or deoxynucleotide triphosphates; dATP, dCTP, dGTP, dTTP).

DNA polymerase: An enzyme that catalyzes the synthesis of new DNA strands by adding nucleotides to a template strand

• The most common are Taq (from Thermis aquaticus), and Pfu DNA polymerase (from Pyrococcus furiosus).

• both can generate new strands of DNA using a DNA template and primers, and are heat resistant

Cofactors: Magnesium ions needed for the proper functioning of the enzyme.

Thermal cycler: a machine programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis (see figure on next page).

steps of PCR

denaturation → annealing → elongatio

denaturation

a segment of DNA is heated so it separates into two pieces of single-stranded DNA

annealing

the binding of primers to the single-stranded DNA templates

elongation

DNA polymerase uses the original strand of DNA as a template to add complimentary dNTPs to the 3’ ends of each primer and generate a section of double-stranded DNA in the region of the

product of PCR

The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment

limitations of PCR

• highly sensitive: any contamination or trace amounts of DNA can produce misleading results

• can only be used to identify the presence or absence of a known pathogen or gene 

• primers may anneal to DNA sequences similar to the target DNA

• infrequently, incorrect nucleotides can be incorporated into the PCR sequence by the DNA polymerase

electrophoresis

the movement of charged particles in solution under the influence of an electric field

commonly used to separate nucleic acids or proteins on the basis of size, electrical charge, shape, and other physical properties.

agarose

a polysaccharide extracted from seaweed and used in gel electrophoresis

current travels:

cathode to anode

cathode

negative electrode

anode

positive electrode

DNA has a ___ charge at neutral pH

negative — which is why they move towards the positive charge in gel electrophoresis!

what size particles go farther in gel electrophoresis?

small particles move faster and farther, while large particles move more slowly and not as far

• Agarose is almost sponge-like in composition, so it’s easier for smaller molecules to get through

loading dye

1) usually contains glycerol which will cause the DNA to sink in the well (rather than float away)

2) It contains a mixture of colored dyes which allow you to track the progress of the gel electrophoresis.

BLAST

Basic Local Alignment Search Tool — a suite of programs used to generate alignments between query and subject protein sequences

expect value

the number of times that an alignment is due to chance

Increased expect value = less reliable alignment (more likely due to chance)

reversion frequency

# revertant colonies per mL/ total CFUs/mL of overnight culture

chromosomal vs. extrachromosomal DNA

chromosomal:

• contains essential genes

• double stranded

• linear or circular

• found in nucleus or nucleoid

extrachromosomal:

• Plasmid 

  • circular, double stranded

  • contain non-essential beneficial genes for the cell (i.e., antibiotic resistance)

the boiling method

% agarose

• determines the separation resolution

• A higher percentage of agarose will resolve smaller DNA molecules and vice versa

true reversion

wild type → mutation by insertion → reversion via removal of the original insertion

second-site reversion

wild type → mutation by insertion → reversion by removal of another base

IFRM structure

implications, findings, results, and methodology

primary source

a source written/composed by the person/people/group who carried out the experiment(s) and/or research that will be explained in the piece