CHEM 153L Midterm+Final Learning Objectives

Fossil fuels

a fuel such as coal, oil, or natural gas formed in the earth from plant or animal remains; a finite resource. Huge contributor to carbon emissions.

Biofuels

generated by treating fresh biomass. The energy is stored as chemical energy in the main storage forms starch or sucrose previously converted by light energy through photosynthesis; from these the microbes/plants/animals can convert this stored chemical energy into biofuels via transesterification or fermentation. They are easy to regenerate.

First generation biofuels

uses food crops for feedstock, leading to competition within the food industry and increased food prices.

Second generation biofuels

uses waste plant material as feedstock, avoiding competition with food production.

Third generation biofuels

uses algal cells or other microbes grown in bioreactors to generate biofuels, which do not compete with agricultural resources and require less land. Some microbes use feedstock, others utilize photosynthesis

Pathway of isobutanol production

involves the overexpression of isobutanol production pathway by hitchhiking on the valine biosynthesis route.

Sucrose

comprised of glucose and fructose

Transmittance

the amount of light that passes through a sample. It decreases exponentially when the sample concentration increases

Absorbance

the amount of light absorbed by a sample. This has a linear relationship with concentration.

Accuracy

how close a measurement is to the reference data.

Precision

the closeness of repeated measurements to each other.

Starch/Amylose

storage molecule of glucose connected by 1,4 glycosidic bonds. Amylopectin aka the 1,6 glycosidic bonds create branching

Glycogen

glucose storage molecule with lots of branching from 1,6 glycosidic bonds

What kind of molecule is isobutanol?

alcohol

What is the advantage of isobutanol as a biofuel molecule over ethanol?

Isobutanol has a higher energy density than ethanol because isobutanol has more carbons. Additionally, isobutanol is less miscible with water than ethanol. When you mix ethanol, gasoline, and water to transport the gasoline, some of the ethanol converts into water, thus some of the gasoline volume is lost. This does not happen with isobutanol.

How can microorganisms be utilized to generate ethanol?

Ethanol can be produced via anaerobic respiration called fermentation. This occurs because during glycolysis, NAD+ is converted to NADH and is depleted and TCC can’t be utilized. Bacteria can also be genetically engineered to perform fermentation in aerobic conditions as well.

Why does the pathlength of samples vary in our lab?

When using a cuvette spectrophotometer, the light is directed horizontally so the pathlength will be the same for all samples so long as the same cuvette is used. While using a microplate reader, the light is directed through the well top to bottom so it is dependent on the volume of the sample loaded into the well.

NAD+ ______ absorb light at 340 nm

does not

NADH ______ absorb light at 340 nm

does

NAD+ to NADH is

a reduction

Describe the hydride transfer that happens during the alcohol dehydrogenase reaction

Zinc in the active site of the enzyme polarizes the carbonyl group of the aldehyde molecule thus stabilizing it. This allows the hydride transfer from NADH to acetylaldehyde. This produces NAD+ and ethanol.

1 assumption of the Michaelis Menten Equation

[ES] stays constant since the rate of formation is the same as the rate of consumption of [ES]

When [substrate] is much below Km,

rate will be low and therefore more dependent on the [substrate]

When [substrate] is at Km,

then the reaction rate is at ½ Vmax

When [substrate] is much above Km,

the rate will be close to Vmax

Alcohol dehydrogenases have

a broad substrate specificity. They also prefer either NADP+ or NAD+. YqhD prefers NADP+

Why do we need to find optimal pH of an enzyme?

Depending on the pH , specific amino acids may be protonated or deprotonated. For the enzyme to be efficient, we need them to be at a specific protonation state. Different pHs can also affect protein folding thus the enzyme does not work.

Why do we need to find optimal temperature of an enzyme?

increasing the temperature increases the activity. However, if the temperature is too high, the protein can denature.

First order reaction

the reaction rate is dependent on concentration of one reactant. Thus, increasing the substrate increases the reaction rate

Zero order reaction

the reaction rate is dependent on the enzyme concentration. Maximum amount of substrate and optimal pH and temperature

At lower substrate concentration, the reaction is at ______ order

first

At higher substrate concentrations, the reaction is ____ order

zero

What is the Michaelis Menten graph plotting?

substrate concentration vs. reaction rate

Km

the substrate concentration at which half of the active sites have substrate bound and the reaction catalyzes. It is a measure of an enzyme’s affinity for its substrate

the lower the Km, the _______ affinity for the substrate

higher

How will we estimate Vmax and Km from the lab?

Using the Michaelis Menten graph, we can guess where the Vmax is and where half the Vmax is to find Km just by looking at the graph. We can also use the raw data and nonlinear least squares analysis to get an exact estimate.

How will we analyze Vmax and Km from the lab?

We will use the Michaelis Menten equation and graph to make a Lineweaver-Burke plot, and we also use non-linear regression analysis

How will we calculate Vmax and Km from the lab?

using non-linear regression and using the equation of the lineweaver burke line

Specific Enzyme Activity

Measures how many µmol of product per minute that 1 mg of enzyme produces under optimal conditions

Is every point on the Michaelis Menten graph an initial rate?

Yes, the only thing that varies is the substrate concentration

Why do we care about 1st order kinetics?

in order to characterize enzyme activity, we like to see how little substrate we can give the enzyme for it to get up to speed. This allows us to talk about the enzyme’s affinity for the substrate

In the lab, why must we be worried about the initial rate?

We are measuring the product concentration, thus we need to see the most accurate representation of the product concentration. This means that the backwards reaction has not kicked in yet and the product has not converted back to substrate.

Why is the Vmax alone not meaningful?

Vmax depends on how much enzyme is present in the reaction. More enzyme means more product which means that the Vmax is faster.

Turnover Number

Measures how many µmol of product per minute that 1 µmol of enzyme can produce under optimal conditions. This can be calculated using Vmax/total enzyme concentration

Why are bacterial cells beneficial for protein overexpression?

They are cheap to cultivate in large numbers and small space, they grow your POI exponentially, it’s easy to tell them what to make and they’ll make a lot of it, we can add a tag to our POI so we can purify it.

Which growth phase will bacterial cells be most efficient in expressing foreign proteins?

The log phase is most efficient for expressing the protein of interest because this is when the bacteria have already responded to the new medium by transcribing genes to translate enzymes and proteins necessary for growth and division. Thus, they are more likely to take up the plasmid!

What factors affect induction efficiency?

temperature, incubation time, and inducer concentration

How does temperature affect protein induction efficiency?

We know that E. coli grow the fastest at 37 degrees C, but if we grow the cells below the optimal, they will grow slower and express the foreign protein at a slower rate. This is especially true if the protein is problematic for the cell. Thus, slowing down the rate of POI expression will make sure that the protein is folded properly and less toxic to the cells. Thus, decreasing temperature is more efficient for POI expression

How does induction time affect protein induction efficiency?

A longer induction causes more protein to be formed, however, they may not be active so they could build aggregates with each other and form inclusion bodies. If the protein is toxic to the cell, it may be best to have a shorter induction period.

How does inducer concentration affect protein induction efficiency?

more inducer causes more protein formation

Why is it bad for the proteins to form inclusion bodies?

The formation of inclusion bodies causes the protein to not be soluble in solution, which makes it harder to purify.

What is bacterial transformation?

The uptake of foreign DNA by bacteria. Bacteria are incubated in calcium chloride at 0 C and are heat shocked/electroshocked

Why are we growing the E. coli in LB medium?

We are giving the bacteria more than what they need to grow, so we are giving them the optimal conditions to grow.

What is IPTG used for?

It is to induce protein expression through mimicking lactose and triggering transcription of the lac operon, thus allowing the bacteria to take up new genes. It is used often because it cannot be degraded by the cell.

Selectable marker (in our case)

The selectable marker allows us to select for only the bacteria that contain the plasmid to grow. In our case, we are adding ampicillin to our medium. The plasmid contains a beta-lactamase, that cuts that lactam ring of ampicillin. Thus, only cells containing the plasmid will survive.

Why do you need to determine the cell density?

As the inoculation time increases, the amount of cells will increase. If you add equal volumes of culture into each well, the amount of cells in each well will vary. Thus, we don’t know if the increase in protein of interest was due to an increase of cells or due to your cells producing more protein of interest

How can you make sure you are putting the same amount of cells in each well?

We take an optical density measurement at 600 nm and see how much higher the cell density is between samples t1, t2, and t0. We can adjust the volume of resuspended pellets so we can ensure that we load equal amounts of protein in each well.

How can we tell the cell to produce our protein of interest?

plasmids can be used! The cells pick them up during bacterial transformation and we can add a gene that codes for our protein of interest

Origin of Replication

An AT-rich region that is recognized by the helicase so that it can open up the plasmid to replicate before cell division. Without the origin of replication, the bacterium cannot replicate or copy the plasmid.

Why do we have only one type of selectable marker?

when we transform cells with these plasmids, we shock them and only a small percentage of the cells actually pick up the plasmid. This is because the cells don’t want to make our protein of interest because it is not advantageous to them.

Multiple cloning site

Where the gene of interest to be expressed is inserted

Promoter

signal sequence that is recognized and bound tightly by RNAP to initiate the transcription of the gene of interest. RNAP synthesizes an mRNA that is a copy of that.

Transcription termination

signal sequence for RNAP to tell it to stop transcribing

operator

signal sequence that is recognized and bound by repressor protein that then blocks promoter so RNAP cannot bind and to inhibit transcription of gene of interest. This is only turned on when the bacteria is in log phase.

Lac I Gene

gene that encodes for the lac repressor. The lac repressor protein recognizes the operator and binds to it and prevents the expression of the protein of interest. IPTG removes the repressor

Ribosome binding sequence

signal sequence that is transcribed to RNA molecule that is then recognized by ribosomes to start translation sequence. This recognizes mRNA and binds to them

Histidine 6 Tag

sequence that encodes for 6 histidine residues that are fused to the 5’ or 3’ end of the gene of interest to be transcribed into the same mRNA molecule. These are important for being able to purify our protein of interest with affinity chromatography. The YqhD gene precedes this.

What is the natural function of the lac-operon?

This is used to control the expression of genes needed for importing and breaking down lactose. It includes an operator, promoter, regulatory gene, and a repressor. The repressor binds to the operator, thus blocking the promoter so that the RNAP cannot transcribe the gene of interest, thus our protein of interest does not get developed. The repression continues until an inducer is added (IPTG in our case)

How is the lac operon expressed, thus how is our gene of interest expressed?

By adding IPTG, which is a lactose derivative. IPTG binds to the active repressor, which leads to a conformation change that decreases the binding affinity of the repressor to the operator. Now the promoter is accessible for binding RNAP and the gene of interest is transcribed in mRNA and then the mRNA is translated into our protein of interest.

What does SDS PAGE stand for

sodium dodecyl sulfate polyacrylamide gel electrophoresis

What is an SDS page used for?

It is used to separate proteins by their molecular weight via an electric field. We load a ladder that contains a mix of proteins with known sizes to compare to our protein of interest.

What is the role of using the detergent SDS while simultaneously boiling the sample?

Since all the proteins have different charges and we want them to run down the gel, we use SDS to make all of the proteins negatively charged, thus they have a uniform charge/mass ratio. Additionally, we must boil the sample with SDS so that we can disrupt the secondary, tertiary, or quarternary structure of the proteins. If they are not denatured, the different shapes can affect how they run through the gel (and not just their molecular weight is a factor to where they end up). Denaturing causes them to linearize.

What kinds of bonds cannot be disrupted by treatment with SDS?

Covalent bonds, specifically disulfide bridges. They require a reucing agent to be broken.

What is the role of adding B-mercaptoethanol or dithiothreitol to the loading buffer?

Both of these are reducing agents that break disulfide bridges

What is the role of adding bromophenol blue to the mix?

The polyacrylamide gel, the electrode buffer, and the proteins are all clear. Thus, we add this dye that does not bind to the proteins to observe the process of the electrophoresis run and know when to turn off the run. The tracking dye runs through the electric field ahead of your proteins like a small protein.

What are the materials in the gel itself?

The gel is comprised of acrylamide and bisacrylamide. Acrylamide alone will form linear polymers called polyacrylamide via a radical polymerization reaction. This creates a polymer that is not very stable. We need a stable gel because we are moving the gels around a lot. Thus, we add bisacrylamide, which can be radicalized at both ends and connect two linear polymers. This ensures stability of the gel.

How do the materials of the gel relate to pore size?

To adjust pore size between polymers, we can adjust the concentration of either acrylamide or bisacrylamide. Increasing the concentration of acrylamide (alone) causes smaller pores, while decreasing the concentration causes larger pores. We can also adjust the concentration ratio of acrylamide and bisacrylamide. Larger pores comes from increasing the ratio, smaller pores come from decreasing the ratio (increasing bisacrylamide concentration)

What is needed to initiate the polymerization reaction of acrylamide/bisacrylamide monomers?

We need radicals to start the polymerization reaction. We use ammonium persulfate as a radical indicator, but we need APS and TEMED together. TRIS is also added to stabilize the pH of the gel.

What is the purpose of Coomassie blue vs the purpose of bromophenol blue?

Coomassie blue is used for protein staining after SDS PAGE and bromophenol blue is used as a migration front indicator (and pH indicator too) during running

What is the stacking effect?

The utilization of glycine zwitterions and chloride ions to ensure all the proteins start running down the gel from the same starting line. This is due to changes in pH causing an increase in voltage from resistance.

Why are gradient gels useful?

They separate both small and high molecular weight proteins from ones similar in their respective sizes in one gel. At the bottom, the pores are the smallest and at the top, the pores are the largest. This ensures that proteins of different sizes are separated well.

Why is it important to add a pH buffer in the gel?

The buffer stabilizes the pH values between neutral and a pH of 9. The correct pH is necessary to ensure that glycine is in the correct protonation state for the respective part of the gel.

What pH buffer is used in the gel?

TRIS

What would happen in a gel run without the stacking gel?

The bands on the gel would not be crisp and focused thus the proteins just run on the gel.

What is the stacking effect dependent on?

The large pores in the stacking gel that don’t hinder any proteins to run.

What are the pHs for the stacking and separating gels?

There is a pH gradient between the two: the stacking gel has a pH of 6.8 where glycine becomes a zwitterion after being protonated and the separting gel has a pH of 8.8 where glycine will become deprotonated and migrate to the bottom of the gel, thus stopping the stacking effect. This allows proteins to separate by molecular weight.

Explain the effect of zwitterions in the stacking gel.

Before glycine interacts with the gel, it has a negative charge (it exists in the running buffer). Once it interacts with the gel, though, it becomes protonated and thus zwitterionic. Thus, the glycine does not move down the gel and creates resistance. This resistance causes the voltage to increase which pushes the proteins towards the top of the stacking gel to be pushed down to where the separating gel starts.

What is the structure of the SDS-page gel?

The negative electrode running buffer with a pH of 8.3 to 8.5 sits towards the top and consists of TRIS and negatively charged glycine. Next is the stacking gel which has TRIS and chloride ions, and this has a pH of 6.8. Then we have the separating gel which has a pH of 8.8. Then, we have the positive electrode buffer, which has the same pH as the negative electrode buffer.

What would happen if the pH of the running buffer was not correct? What if it were instead set to a pH of 6?

If the running buffer had a pH of 6, the glycine would stay as zwitterions and they would not move down the electric field. All of the proteins would run on the gel, but they would not be in focused bands because glycine would not enter the stacking gel and thus insufficient stacking occurs.

What is the purpose of the protein standard ladder?

it is used to estimate the molecular weights of proteins separated by SDS PAGE

Affinity

The larger the free energy change when two molecules bind, the stronger the interaction. This can be measured by the dissociation constant

How does CBB bind to proteins?

When CBB is at a pH above 2, the overall charge is negative, which causes the blue color and this is when it has a high affinity for proteins. It has the highest affinity for arginine, but will also bind to His, Lys, Phe, Trp, and Tyr. It does not bind to negatively charged side chains.

Specificity

this describes the preference of a molecule to bind to one particular target relative to all others. We can compare the dissociation constants of this molecule binding to other molecules. If the Kd is lower compared to all others, the affinity and the specificity is high. If the Kds are similar, the specificity is low

How do hydrophobic amino acids affect specificity and affinity?

decrease specificity, but increase affinity since they can make a hydrophobic contact surface

How do polar/charged amino acids affect specificity and affinity?

increase specificity because they need the complementary sidechain on the binding partner in the exact position. They also increase affinity.

How do we transfer the proteins from the SDS PAGE onto a nitrocellulose membrane?

We use a horizontal electric field and put the nitrocellulose membrane on the positive electrode so that the proteins bind to the membrane after leaving the gel. This is called a horizontal transfer. We also add ethanol to the transfer buffer to strip SDS from the proteins during transfer to enhance protein binding to the membrane.

Why don’t we put antibodies directly onto the gel?

Antibodies are relatively large proteins and it would take forever for them to run down the gel and find the His6 tagged proteins.

What would an immunoblot look like if blocking was forgotten?

Antibody molecules will bind to the nitrocellulose membrane since the membrane has a high affinity for all proteins. Thus, the signal (color or light) will be detected non-specifically all over the membrane during detection.

How can you minimize nonspecific protein protein interactions?

This can be prevented by incubating the membrane in high salt concentrations which only allows for specific interactions to occur. This is why we use TTBS. Adding a mild detergent at a low concentration has a similar effect and is also included.

How can you minimize nonspecific protein surface interactions?

Blocking with casein inhibits non-specific protein surface interactions by covering all the gaps between protein bands. This minimizes background signals