BIOCHEM 153L Study Guide

What are first-generation biofuels?

use food crops to produce biofuels. Directly competes with the food industry, thus impacting prices of staple food. The digestion of feedstock is simple.

What are second-generation biofuels?

use plant waste to undergo anaerobic fermentation to produce biofuels. Does not compete with the food industry but the biomass is in the cell walls (cellulose, hemicellulose, lignin) so this is harder to digest.

What are third-generation biofuels?

engineering algae to produce biofuels. They directly capture atmospheric CO2. These can be grown in illuminated bioreactors on land that is useless for agriculture thus it does not compete with the food industry and is easy to grow.

What is sucrose?

a disaccharide of glucose and fructose covalently bonded

Glycogen vs starch

-starch is the storage form of glucose in plants while glycogen is the store form of glucose in animals

-glycogen is more branched than starch

What is the most abundant biomolecule on the planet?

cellulose

What are the advantages of using isobutanol over ethanol for biofuels?

higher energy density and is less miscible with water

How does glucose become converted to isobutanol?

glucose -> pyruvate -> 2-acetolactate -> 2,3-dihydroxy-isovalerate -> 2-ketoisovalerate -> isobutyraldehyde -> isobutanolWhat

What reaction does acetolactate synthase catalyze?

pyruvate -> 2-acetolactate with CO2 released

What reaction does acetohydroxy acid isomeroreductase catalyze?

-reduction reaction

2-acetolactate + NADPH-> 2,3-dihydroxy-isovalerate + NADP+

What reaction does dihydroxy-acid dehydratase catalyze?

2,3 dihydroxy-isovalerate -> 2-ketoisovalerate

What reaction does 2-ketoacid decarboxylase catalyze?

2-ketoisovalerate -> isobutyraldehyde with the release of CO2

What reaction does alcohol dehydrogenase catalyze?

isobutyraldehyde + NADPH -> isobutanol + NADP+

How do you calculate transmittance from intensity?

T= intensity after it has gone through the sample/initial light intensity

How are absorbance and transmittance related?

A=-logT

What is the relationship between absorbance and concentration in the sample?

linear

What is the relationship between transmittance and concentration in the sample?

exponential

Does NAD+ or NADH absorb light at 340 nm?

NADH

What ion is in the active site of the alcohol dehydrogenase enzyme?

Zn2+

Does YqhD have a preference for NADPH/NADP+ or NADH/NAD+?

NADP+/NADPH

Is aldehyde -> alcohol a reduction or oxidation reaction?

reduction

What are the "optimal testing conditions" for an enzyme?

substrate availability, temperature, pH

What is the steady state assumption?

rate of ES formation = rate of ES breakdown

What does Km represent?

-Substrate concentration at 1/2 Vmax

-indicates affinity of enzyme for substrate

What is first order reaction and how does it appear on a Michaelis Menten graph?

-reaction rate is dependent on concentration of one reactant

-appears at the beginning of a michaelis menten graph when the concentration of substrate is low

What is zero order and how does it appear on a Michaelis Menten graph?

-reaction rate is independent of substrate concentration but rather on enzyme concentration

-appears near Vmax in a michaelis menten graph

How do you calculate Vmax?

-enzyme concentration in molarity X Kcat OR enzyme concentration (mg/mL) X enzyme activity

What does Kcat measure?

it is the turnover number that measures micromoles of product per minute formed that 1 mg of micromole of enzyme can produce

What does specific enzyme activity measure?

it is the amount of micromoles of product formed per minute by 1 mg of enzyme

Why is it important to vary the concentration of the enzyme?

the concentration of the enzyme can effect the speed of the reaction and we want to keep our reaction within initial conditions so it is important that we test different concentrations

During which growth phase will the cells be the most efficient in expressing foreign proteins?

log (exponential) phase because this is when they are displaying the greatest metabolic activity and likely to have the most ribosomes per cell

Why would it not be best to induce cell cultures in the lag phase?

the cells need a bit of time to sense the changing environment and space and begin transcribing genes to translate enzymes and proteins necessary for growth and division

What factors influence protein induction efficiency?

concentration of the inducer, duration of IPTG, and temperature

What is the purpose of a selectable marker in a plasmid?

Selectable markers are genes that help identify bacteria that have successfully transformed, or taken up the recombinant plasmid

What is the purpose of the origin of replication in a plasmid? What would happen if our plasmid did not have an origin of replication?

-it is where replication machinery will recognize the location to start replicating the DNA

-without the origin of replication, the plasmid would not be replicated along with our POI

What is the purpose of the LacI gene in the plasmid?

it encodes for the repressor protein which interacts with the lac operator and keeps the expression of the his6-yqhD off; this is important because we do not want to induce the expression of the protein until the cells are in the log phase

What is the purpose of the bacteriophage promoter in the plasmid?

this is the site where RNA polymerase recognizes the DNA and binds to initiate transcription of a gene

What is the purpose of the operator (lac O) in the plasmid?

it is located close to the promoter and when the lac repressor binds to the operator, the promoter is no longer accessible to RNA polymerase and the gene cannot be transcribed

What is the purpose of a multiple cloning site?

it is a series of restriction enzyme recognition sequences which allows for one to insert the gene of interest

What do the histidine residues bind to in the affinity chromatography column?

they bind to nickel

Why is IPTG used as an inducer of lac promoter activity?

it will not be metabolized or degraded by the cell because it is a gratuitous inducer while allolactose is a natural inducer which will be metabolized

What does SDS PAGE stand for?

sodium dodecyl sulfate polyacrylamide gel electrophoresis

How does SDS PAGE separate proteins?

based on molecular weight only

How are proteins separated in SDS-PAGE (what kind of field is induced)?

electric field where the bottom of the gel has a positive electrode and the top has a negative electrode

What are the two compounds that are used to get all proteins negatively charged in SDS-PAGE?

SDS and B-mercaptoethanol

How is SDS used in SDS-PAGE and what does it do?

it is a detergent called sodium dodecyl sulfate that is used with simultaneous boiling of the solution

-it breaks non-covalent bonds that stabilize secondary, tertiary, or quaternary structures

-it also covers proteins with a negative charge, approximately 1 negative charge per 2 amino acids

What is the purpose of a reducing agent in SDS-PAGE?

it breaks disulfide bridges that may be present in proteins

What are the two reducing agents that can be used?

DTT (dithiotreitol) or B-mercaptoethanol

How do we monitor the progress of the SDS-PAGE?

bromophenol blue tracking dye

How is pore size affected by the ratio of acrylamide/bisacrylamide?

higher ratio-> larger pores

lower ratio-> smaller pores

What are the two components of the radical polymerization reaction that occurs in the SDS page gel?

acrylamide and bisacrylamide

How does the radical initiation of the polymerization reaction of acrylamide/bisacrylamide monomers occur?

addition of ammonium persulfate and TEMED (tetramethylethylenediamine)

What is the purpose of TRIS buffer in SDS PAGE?

it is a pH buffer that ensures glycine is in the correct protonate state for the respective part of the gel

What is the running electrode buffer that is used in SDS-PAGE and what is its purpose?

TRIS buffer- keeps glycine molecules in its negative state so that they will run through the gel

What is the pH difference between the stacking and separating gels in SDS-PAGE?

-the stacking gel has a lower pH of 6.8 so that the glycine molecules become zwitterions

-the separating gel has a higher pH of 8.8 so that glycine resorts back to being negative which can run to the positive electrode and allow proteins to separate by their molecular weight

How does the stacking gel work?

-the glycine zwitterions are neutrally charged which creates resistance while negative chlorine ions are the leading ions that move with low resistance and therefore low local voltage > these proteins closer to the leading ions will run slower than the proteins further up

-the proteins can therefore be compressed to a single line before entering the separating gel

What would happen if you set the pH of the running buffer to pH 6 instead of 8?

glycine would not enter the stacking gel in large numbers (would already be in zwitterion state) and therefore the stacking effect would be less dramatic -> we wouldn't see focused bands

What is the relative size of the pores in the stacking gel?

they are relatively large so that proteins of all sizes move easily through the pores of the stacking gel

How do we know if normalization occurred in SDS-PAGE?

if the background pattern of proteins looks similar in the gel after adding Coomassie, then the protein samples were likely normalized

How can we test if a protein is pure in solution or if other proteins are in solution as well?

Coomassie staining

What are the three states of coomassie brilliant blue dye?

+1 charge: red form which exists at low pHs

neutral charge: doesn't bind to proteins with high affinity

-1 charge: looks visibly blue and absorbs light at 595 nm

Which form of Coomassie brilliant blue binds to proteins with high affinity?

blue- absorbs light at 595 nm

What kind of amino acid side chain can CBB bind to?

The dyes bind via electrostatic interaction with protonated basic amino acids (lysine, arginine, and histidine) and by hydrophobic associations with aromatic residues

Where does the secondary antibody bind to on the primary antibody?

it binds to the constant region of the primary antibody

What is a visualization method for just seeing your POI?

immunoblotting aka western blotting

What is the role of the variable region in an antibody?

this is what binds to the specific antigen

What does the Kd for a ligand and protein represent?

-it gives us a measure of binding strength

-the smaller the Kd value, the greater the binding affinity and more tightly the two molecules interact

Why do we add ethanol when transferring proteins from an SDS PAGE to a porous membrane?

it helps to transfer the proteins from the SDS PAGE gel to the membrane

What compound is the porous membrane covered with?

nitrocellulose

What are the charges of the SDS page vs the porous membrane?

-the SDS PAGE has a negative charge

-the porous membrane has a positive charge

Why is blocking the membrane an important step and what compound is used to do this?

-blocking the membrane is important because it prevents antibodies (which are proteins themselves) from sticking to the free membrane non-specifically (this would cause a signal to be detected all over the membrane during detection)

-casein or BSA are used as the blocking agent

How do antibodies allow us to only obtain our POI?

it uses an antibody that binds to protein of interest with high specificity and affinity

What is the antigen in the case of immunoblotting?

POI

Why do we often use a secondary antibody for immunoblotting?

using a secondary antibody conjugated to an enzyme or dye molecule ->signal amplification -> more sensitive detection

What is the difference between an antigen and epitope?

-antigen -> molecule that the antibody binds to via the variable region

-epitope -> the part of the antigen that the antibody directly binds to (binding site)

What are non-specific protein-surface interactions?

this occurs when the antibody binds to the nitrocellulose directly independent of the location of the antigen (POI)

What are non-specific protein-protein interactions?

this occurs when the antibody binds to other proteins on the nitrocellulose independent of the location of the antigen (POI)

How can the frequency of non-specific protein-surface interactions be decreased?

using blocking agents like milk protein casein or BSA

How can the frequency of non-specific protein-protein interactions be decreased?

-adding low concentrations of mild detergent

-incubating the membrane in high salt concentrations to allow for only specific interactions to occur

What is a disadvantage of using colorimetric detection for visualizing immunoblots?

low sensitivity (you need a lot of antigen for this to work properly)

What is colorimetric detection with immunoblotting?

this is where the enzyme attached to the antibody catalyzes a reaction that forms a colored precipitate product from a colorless substrate

What is chemiluminescent detection with immunoblotting?

enzyme attached to the antibody catalyzes a reaction that forms an unstable product which decays and emits light at a certain wavelength

What is fluorescent detection with immunoblotting?

a fluorescent dye is conjugated to a secondary antibody and can be excited by a specific wavelength and emit light (no enzyme needed)

What enzyme do we use for the colorimetric detection with immunoblotting?

alkaline phosphatase

What reaction does alkaline phosphatase catalyze?

NBT and X-P -> salt that gives off a purple color

What is NBT and X-P? When did we use them in our lab course?

Nitroblue tetrazolium and 5-bromo-4-chloro-3-indoylyl phosphate; these were the reactants for a reaction catalyzed by alkaline phosphatase- colorimetric detection with immunoblotting

What is a potential benefit of using the chemiluminescent method over colorimetric

more sensitive and doesn't take as much of the target protein

What is the difference between monoclonal and polyclonal antibodies?

-monoclonal antibodies only recognize a single epitope per antigen and are therefore highly specific

-in contrast, polyclonal antibodies consist of a heterogeneous mixture of antibodies, with each antibody recognizing different epitopes of a particular antigen

What is imidazole?

it is the side chain of histidine

What is the purpose of Bugbuster solution and when did we use this in our lab?

-it has mild detergents that will disrupt cell membranes with lysozyme which will degrade the bacterial cell walls

-we used this in the purification step of His6::YqhD

What are inclusion bodies?

this is when the POI (usually as a result of strong overexpression) aggregates and becomes insoluble

What is the purpose of centrifugation after cell lysis?

it makes an insoluble pellet and soluble supernatant

How can we ensure that the proteins are stabilized?

-pH conditions, temperature, inhibition of proteases, retardation of microbes that can destroy proteins

What is the purpose of a spacer in affinity chromatography?

makes it more available to a ligand and less restricted by steric hindrance by the matrix

What is the ligand in our affinity chromatography?

Ni-NTA

How do we equilibrate the ligand in affinity chromatography?

we add low amounts of imidazole to prevent proteins with single histidine residues from binding to the matrix

Why do we use increasing concentrations of imidazole to the affinity chromatography column?

-we use the lowest concentration to equilibrate the ligand

-then we use higher concentrations in order to outcompete weak bindings for proteins that got stuck to the column specifically

-the highest concentration is used to elute the his-tagged protein which can be outcompeted by the imidazole

What enzymes and components did the first plasmid (pSA69) have?

-it had the lac operator, promoter, selectable marker for kanamycin resistance, origin of replication, acetolactate synthase, acetohydroxy acid isomeroreductase, and dihydroxy acid dehydratase

-this plasmid was received by all of the strains besides the control

What enzymes and components did the second plasmid have?

-it had the lac operator, promoter, ampicillin resistance, origin of replication, ketoacid decarboxylase, and differing alcohol dehydrogenases

-this plasmid differed for all of the strains

How is kanamycin toxic to bacterial cells?

it interferes with protein translation