Biomed Lab I FINAL

Prokaryotes

organisms made up of cells without a nucleus or any membrane-encased organelles

ex: bacteria

Plasmid

small, circular extrachromosomal DNA found in bacteria

Genome

the complete set of genetic information of an organism

Chromosomes

long strands of DNA molecules that store an organism’s genome

Genes

short segments of DNA containing protein-coding information

• located in chromosomal DNA

DNA

• aka deoxyribonucleic acid

• long chains composed of repeating subunits called nucleotides

  • Adenine, Tyrosine, Cytosine, Guanine

• antiparallel strands

  • DNA 5’ … 3’

  • RNA 3’ … 5’

pGLO Plasmid

recombinant pGLO plasmid that contains several genes and DNA sequences that enable replication of the plasmid DNA, differentiate and re pGLO transformed bacteria

GFP

a jellyfish gene that codes green fluorescent protein; responsible for green fluorescent phenotype

pBAD Promoter

a specific DNA sequence upstream from the GFP gene, which binds to araC-arabinose and promotes RNA polymerase binding and transcription of GFP

bla

a gene that encodes the enzyme beta-lactamase

• beta-lactamase breaks down the antibiotic ampicillin

responsible for antibiotic resistant phenotype

ori

the origin of pGLO plasmid DNA replication

araC gene

a gene that encodes the regulatory protein that binds to the pBAD promoter

only when arabinose binds to the araC protein is the production of GFP switched on

araC Protein

regulatory protein encoded by the araC gene

binds to the pBAD promoter

Arabinose

a sugar that binds to the pBAD promoter and displaces the acaC protein from pBAD promoter so RNA polymerase can bind and initiate transcription

only when arabinose binds to araC protein is the production of GFP switched on

Ampicillin

an antibiotic that kills bacteria by destroying beta-lactamase

Recombinant DNA Technology

a method used to modify the gentic properties of an organism

useful in preparing proteins that are used in research and therapy

Steps of Recombinant DNA Technology

1) Synthesis of a recombinant plasmid

• genes are cut & recombined to create a plasmid

• plasmid must contain gene of interest, DNA for replication sequence, antibiotic resistance (may also have regulating & transcription gene

2) Transformation of the organism

• plasmid is inserted into bacteria cell

3) Selection of the transformed organism

• transformed cell is grown in antibiotic-containing media to allow plasmid to grow

4) Synthesizing the gene product (protein of interest)

• cells are grown in media that contain molecules required for producing protein

Competent Cells

cells are capable of taking in foreign DNA

positive charge is used to treat cells to allow DNA to enter cell through cell membrane

Bacterial Transformation

method used to introduce plasmid DNA in bacteria

bacteria is made competent to take up foreign negatively charged DNA

QIAprep Spin Miniprep Kit

Buffer P1

Buffer P2

Buffer N3

Buffer PE

Distilled Water

Buffer P1

Resuspension Buffer

• removal of ions weakens cell membrane for lyse

• solution looks cloudy

Buffer P2

Alkaline Lysis Buffer

• ruptures cells and denatures DNA into single strands

  • solution looks clear to show lysis of cells & solubilation of cellular components

Buffer N3

Neutralization Buffer

• neutralization allows DNA to renature

• solution appears milky

Buffer PE

Wash Buffer

• ethanol helps wash chaotropic salts & other contaminants (buffer removed by centrifuge)

Distilled Water

water recovers “hydration shells” around plasmid DNA & dislodges plasmid DNA from silica gel membrane

Mutation

a change in nucleotide sequence of short region of genome, resulting in changes that may lead to disease

Silent Mutation

mutations that do not show a change in translated amino acid, hence no change in phenotype

Polymorphism

naturally occurring genetic differences among organisms in the same species that does not lead to disease

Beer-Lambert’s Law in Relation to Nucleic Acid Quantification

A = εbc

A = absorbance (AU)

ε = absorptivity coefficient for extinction coefficient ((ug/mL)^-1cm^-1)

b = light path (cm)

c = concentration (ug/mL)

Average Extinction Coefficient for DNA

0.020 (ug/mL)^-1cm^-1

Average Extinction Coefficient for RNA

0.025 (ug/mL)^-1cm^-1

Nucleic Acid Purity for 260/280 Ratio

1.8-2 Pure DNA

<1.8 Presence of proteins, alcohol, acidic pH, other contaminants

>2 RNA contamination, basic pH

Nucleic Acid Purity for 260/230 Ratio

2.0-2.2 Pure DNA

<2 EDTA, carbohydrates & phenol

Palindromic Sequence

a segment in DNA or RNA that is the same sequence when read from 5’ to 3’ on one strand and 5’ to 3’ on the complementary strand

Restriction Enzymes

EcoRI

Hind III

Codon

a three-nucleotide sequence of DNA that codes for a particular amino acid

different codons can translate to the same amino acid, leading to silent mutations

Restriction Digest as a Tool to Detect Mutagenesis

when there is a silent mutation within a restriction enzyme’s restriction site, the mutated sequence will not be cut by the enzyme

DNA Shapes

Supercoiled DNA

Linear DNA

Nicked DNA

UV vs Nanodrop Spectrophotometer

UV uses larger sample sizes

Nanodrop uses smaller sample sizes

Protein Structure

Proteins are made up of amino acids linked by peptide bonds

AA Sequence of GFP

serine-tyrosine-glycine

Gene Expression

entire process from transcription through protein synthesis

Gene Regulation

process that allows mRNA and protein synthesis only when and where the encoded protein is required

Promoter

DNA sequence upstream to the coding region of a gene to which the RNA polymerase binds to initiate transcription

Regulatory Molecules

activates or inhibits gene transcription

Gel Electrophoresis

procedure used to separate biomolecules using a gel matrix based on size using electric currents

larger molecules migrate more slowly than small

Gel Matrix

contains pores of uniform size through which molecules can pass through

the smaller the pores, the higher the concentration of the gel

SDS PAGE

made up of polyacrylamide gel used to separate proteins by molecule size

Laemmli Buffer dissociates proteins into individual polypeptide subunits

Sodium Dodecyl Sulfate (SDS) denatures protein complexes (and added to running buffer to mask charges)

Dithiothreitol (DTT) cleaves disulfide bonds

Protein Marker

mixture of proteins of known molecular weight that is loaded on the gel to estimate the size of proteins in an unknown sample

Total Protein Estimation

quantified (assayed) using Coomassi blue dye

arginine and nonpolar AA residues interact with the dye causing a shift in dye absorption max, from 465 to 595nm to give blue color

intensity of blue is proportional to protein concentration

Bovine Serum Albumin

commonly used for generating a standard curve

DNA Marker Example

Lambda DNA digested with Hind III enzyme

Amino Acid Functional Groups

Carboxyl group (COOH)

Amino group (NH2)

Isoelectric Point

the pH at which a protein carries no charge

• below, proteins carry net positive charge

• above, protein carry net negative charge

Chromatography

procedure used to separate individual compounds present in a mixture based on the different physical and chemical properties and determine relative concentration of the compound in mixture

Stationary Phase

thin layer on solid support where compounds are separated based on their relative affinity to the stationary and mobile phase

• if compound has greater affinity for mobile phase, compound will move with mobile phase

• each compound in sample moves at different speeds through stationary phase

Normal Phase Chromatography

relatively more polar stationary phase and usually made of silica gel

• hydrophobic compounds will move quickly

• silica gel is acidic and offers poor separation of basic samples, causing deterioration

Reversed Phase Chromatography

relatively more nonpolar in stationary phase due to matrix particles (silica gel) being coated with hydrophobic chains (C18 chains)

Mobile Phase

can be liquid or gas that moved through the stationary phase via capillary action or with the use of pressure

Gel Electrophoresis vs Chromatography

Gel separates based on molecule size

Chromatography separates based on polarity

Size Exclusion Chromatography

SEC separates molecules based on size by filtration through gel (consisting of spherical beads with pores of specific size)

• smaller molecules will get trapped in the pores

• larger molecules will flow to the bottom more quickly

Thin Layer Chromatography

technique where sample is spotted onto a stationary phase with the bottom edge is placed in a mobile phase

• mobile phase moves up the stationary phase via capillary action

• compound with greater affinity for mobile phase moves up along with the mobile phase

Retardation Factor

value used to compare movement of individual compounds in sample mixture

Rf = distance traveled by compound / distance traveled by mobile phase

Ninhydrin

color reagent used to convert AA to colored compound (usually purple)

High Performance (or Pressure) Liquid Chromatography

technique that uses a solid stationary phase packed in a column, mobile phase (aka eluent) is pushed through column under constant pressure using pressure pumps

• reverse phase most common in analysis of drugs & body fluids

Chromatograph

recording of absorbance changes

• when compound is eluted, there is an increase in absorbance, peak is recorded

Retention Time

time required for eluting the compound through the column