Biochemistry Lab: Final Exam

Which of the following statements is/are true? 1. All of the answer choices are ways we can use restriction enzymes 2. We use restriction enzymes in expression vectors to insert DNA sequences where they should be 3. We use restriction enzymes to recognize and cleave a specific DNA sequence 4. We use restriction enzymes to cut DNA to confirm they are of the right size

1. All of the answer choices are ways we can use restriction enzymes

Arrange the following events in the proper sequence for gene cloning: 1 = Incorporate gene into bacterial plasmid 2 = Isolate DNA from organism containing desired gene 3 = Transform bacterial cells with recombinant plasmid 4 = Fragment DNA with restriction enzyme

2,4,1,3

Which of the following statements is/are TRUE? 1. When a linear polypeptide chain is folded into 3D structures, this is called the primary structure. 2. Correct! The study of proteins and their functions is called proteomics 3. Correct! The study of proteins often includes the need to quantify the amount of protein available. 4. Cells translate DNA (into mRNA), then transcribe (the mRNA) into proteins.

2. The study of proteins and their functions is called proteomics 3. The study of proteins often includes the need to quantify the amount of protein available.

You are making your standards for analysis by the BCA assay using a standard stock of 2 mg/mL of BSA protein. To make a total of 1000 µL standard for analysis with a final [BSA] concentration of 10.0 µg/mL, what volume (in µL) of the standard stock solution will you need to add to your Bradford assay?

5

At which wavelength is the absorbance maximum when Bradford dye reagent binds protein?

595 nm

Which of the following will have the egfp gene?: (A) Isolated plasmid pEFGPx3-N1 (B) Isolated plasmid pET-41(a) (C) E. coli with pEFGPx3-N1 (D) E. coli with pET-41(a)

A and C only

In the context of gene cloning, what is a vector?

A plasmid used to house a gene of interest to transmit it from one place to another.

A plasmid is:

A small circular DNA

Based on the article by T. Craggs, what is the proposed function of the highly-conserved glutamate-222 residue?

Acts as a general base in the maturation process.

For this lab, which "type/method" of column chromatography will be be using to isolate and purify our GST::EGFP fusion protein?

Affinity

Which of the following will you include in your PCR reaction?

All of the answer choices: DNA polymerase Deoxynucleotides Primers Template DNA

Which of the following fields is/are a part of Bioinformatics?

All of the answers are correct: Genetics Biochemistry Computer Science Molecular Biology

Where is the egfp gene located in the target plasmid?

Between NcoI and NotI cut sites

What is the "dye" compound utilized in the Bradford assay to determine protein concentration? You MUST be specific and provide the full name (no abbreviations)...and please mind your spelling!!!

Coomasie Brilliant Blue G250

Which of the following is your to-do list for Lab 6 (Colony Analysis) this week? Select only the best answer choice. A. Count the number of colonies on each plate. B. Take a picture of your colonies to affix in your lab notebook. C. Isolate pET-TIGER from these colonies that glow. D. Only A and B E. All of the answer choices are on your to do list this lab.

D.

From which organism will we isolate our plasmid DNA?

E. coli

What does ELISA stand for?

Enzyme-Linked ImmunoSorbent Assay

TRUE or FALSE: "Blanking" a spectrophotometer is optional prior to use.

False

TRUE or FALSE: ELISA can only be used for analysis of proteins and/or polypeptides.

False

TRUE or FALSE: Enzymes act as a catalyst by decreasing the activation energy of the reactions, therefore speeding up the reactions and decreasing the free energy exchange of the reaction.

False

TRUE or FALSE: For the purpose of ELISA, a "reporter" enzyme or tag is always tagged (or "labeled) on a secondary antibody.

False

TRUE or FALSE: Hypothetically speaking...we can expect the (larger) GST::EGFP fusion protein will to travel farther into the SDS-PAGE gel relative to the (smaller) cleaved EGFP portion post-enzymatic cleavage.

False

TRUE or FALSE: In similar fashion to DNA gel electrophoresis, samples containing proteins are denatured/treated and given an overall positive charge. During the SDS-PAGE run, this allows the proteins to migrate towards the negative electrode of the gel.

False

TRUE or FALSE: It is the specialized, unique "epitope" region of every antibody that subsequently binds to a unique "paratope" region of a specific antigen.

False

TRUE or FALSE: Larger proteins (>50 kDa) will migrate better on higher-percentage gels (> 8-10%), while lower-percentage gels (< 12-15%) are best suited for smaller proteins (< 50 kDa).

False

TRUE or FALSE: The standard curve for this week's Bradford assay is created by performing a serial dilution using a sample of an unknown concentration in order to generate a best-fit (or "linear regression") line that can be used to determine the concentration of another unknown sample.

False

TRUE or FALSE: Today's lab and in silico exercises will focus on the pEGFPx3-N1 plasmid, similar to Lab 3.

False

TRUE or FALSE: We can use the ratio of Abs465/Abs595 to roughly determine the relative protein purity of your samples.

False

True or False: To obtain the plasmid, we will subject the cells to acidic lysis this week (Lab 2).

False

True or False: When IPTG is present, the lacI gene will bind to the lac promoter and prevent the transcription of egfp gene.

False

Which of the following acronyms related to the pET-41a(+) plasmid is/are NOT correct?

GST (Glucose S Transferase)

What protein is being purified in this week's lab? YOU MUST BE SPECIFIC and MIND YOUR SPELLING (the abbreviation will be sufficient)!!!

GST::EGFP

The elution buffer utilized in lab this week contains a "specific" compound that releases any bound GST::EGFP recombinant protein from the immobilized resin in the column. What is this compound?

Glutathione

After the lysis of a cell culture this week, the large, insoluble cellular components (i.e., membranes) will be removed using which of the following methods below?

High-speed centrifugation

Which of the following triggers transcription of the lac operon?

IPTG

The lacI gene in pET-41a(+) is "constitutively active". Which of the following statements is FALSE regarding the pET expression system?

IPTG binds to the T7 promoter

After addition of the elution buffer and centrifugation, where is your isolated DNA?

Inside the elution buffer (i.e. the eluant)

Imagine you're working in a research lab, and you endeavoring to design an antibody that will bind to a single epitope with minimal non-specific binding. Should your design be based on a monoclonal or polyclonal basis and WHY?

It should be based on a monoclonal basis because they are designed to have very specific binding (antibodies will only bind to one epitope on one antigen). Additionally, because monoclonal designs are very specific, they help to ensure reproducible results. This makes using a monoclonal design worth the high cost compared to a polyclonal design.

Which selectable marker does your target vector carry?

Kanamycin resistance gene

The correct sequential order of steps in the plasmid prep procedure is:

Lyse cells > Bind plasmid to DNA column > Wash > Elute DNA from Column > Quantify DNA

"Fixing" is an important step prior to staining gels. Older, original methods used a "fixing solution" that usually included methanol and acetic acid. What is the importance of the methanol in these types of solutions?

Methanol extracts SDS from the gel so that SDS does not interfere with staining.

The two restriction enzymes that you will use in this lab are:

NotI and NcoI

The experiment calls for 1µL of enzyme for restriction digestion. Which pipette should we use?

P2

In the procedure for this lab, what will you do after the first time you elute DNA using Elution Buffer?

Pipette your eluant directly back onto the center of the same column

"Proteomics" is the study and analysis of an organism's entire __________.

Proteome

In your cloning experiment, what kind of "ends" of DNA pieces will be generated that allow the gene to be cloned into the vector in one direction?

Sticky ends

A popular enzyme tag (or label) used in ELISA is the enzyme HRP. Many ELISA methods (including today's lab) will make use of what substrate molecule that is subsequently oxidized by HRP to produce the colorimetric change?

TMB

In a brief-but-detail paragraph using complete sentences, please compare and contrast the 3 different formats (or types) of ELISA methods discussed in the Lab 12 handout.

The Direct Elisa method is fast, but unfortunately is both expensive and has low sensitivity. It is best for analyzing immune responses. The Indirect Elisa method is cheap and decently sensitive, but it is slow. It is best for measuring the concentration of the total analyte. The Sandwich Elisa method is both slow and expensive, but it is very sensitive. It is best for impure and complex samples.

For this lab, you will determine which of the following?

The effect of temperature on the activity of beta-galactosidase The effect of pH on the activity of beta-galactosidase The activity and specific activity of beta-galactosidase using ONPG

Which of the following statements about the molecular cloning experiment you've performed in lab is FALSE?

The egfp gene was obtained from the pET-41a(+) vector

In today's lab, we will align the EGFP variant against the native GFP molecule, using the SnapGene software. What is the main main purpose behind this step?

To identify specific mutations made to GFP to create EGFP.

What is the function of a primer?

To identify the particular region of DNA to be copied by PCR

TRUE or FALSE: Absorbance is a unit-less measurement.

True

TRUE or FALSE: For the purposes of last-weeks purification and based on the pET-Tiger plasmid map...the GST-tag utilized for purification (which will be removed for this week's lab) is attached to the n-terminus of the EGFP protein.

True

TRUE or FALSE: From a structural perspective, the majority of an antibody's final structure is highly conserved, with the exception of a pair of variable regions on the "arms" of the antibody.

True

TRUE or FALSE: Generally speaking, for most enzymes, an increase in temperature will often correlate to an increase in activity (until an optimum point is reached).

True

TRUE or FALSE: Proteins can be characterized as "amphoteric", meaning they can maintain both positive and/or negative charges in the same molecule under various conditions.

True

TRUE or FALSE: The "over-expression" of a target protein means that the combined steps of transcription/translation (e.g. DNA → protein) are purposefully enhanced.

True

TRUE or FALSE: The GST Bind resin is quite delicate and vortexing will inactivate it!

True

TRUE or FALSE: The chromophore (fluorophore) region of EGFP can be found at the center of the final, beta-barrel structure after folding.

True

TRUE or FALSE: The chromophore (fluorophore) that gives rise to the inherent fluorescence of GFP (and its variants, such as EGFP) is due to the rearrangement and maturation of a tri-peptide sequence that is part of the protein.

True

TRUE or FALSE: The wavelength maximum is a specific frequency of light where energy absorption is greatest and can be identified in an absorption spectrum as a "peak"

True

TRUE or FALSE: We should expect the GST::EGFP recombinant protein to be soluble.

True

True or False: Digestion opens up the plasmid from circular to linear.

True

True or False: If there are more colonies than you can count on the plate, you will report as TNTC, which stands for "Too Numerous To Count."

True

True or False: The Multiple Cloning Site (MCS) is a stretch of DNA sequence where we can insert our gene of interest at a specific target site.

True

True or False: The pET-41a(+) vector has been engineered with the molecular machinery that encourages the bacteria to produce protein.

True

True or False: The thermal cycler (or thermocycler) is used for both plasmid digestion and PCR.

True

True or False: The three steps of polymerase chain reaction (PCR) are denaturation, annealing, and extension.

True

True or False: We use the T7 promoter to drive the production of the egfp protein.

True

Which of the following assays is best considered to be non-destructive AND non-colorimetric?

UV280

In molecular biology research, what is GFP typically used for?

Used as a reporter

How will you visualize the restriction digest cut result?

Via Gel Electrophoresis

The physical region within the enzyme where the substrate binds is called the "__________" site:

active

The ELISA method incorporates a molecule known as an "antigen", with which a specifically selected antibody will interact. In nature, an antigen is:

any molecule that induces an immune response

The UV280 assay is the direct measurement of UV absorption at 280 nm due to the presence of the __________ amino acids.

aromatic

For the purposes of analysis of the SDS-PAGE gel, you will need to calculate the "Rf" value (or the "retention factor/value") for several, specified bands on your gel. This value is calculated as "_________" divided by "_____________".

band travel distance, total length of the gel

What is the FULL NAME of the enzyme are we using for our lab this week? Please mind your spelling, NO abbreviations!!!

beta-galactosidase

Which of the following components found in the SDS Loading Buffer are used to (or capable of) denature a protein's tertiary structure for analysis by SDS-PAGE?

beta-mercaptoethanol, SDS

From the options below, assign the correct, overall "sequence" for the essential steps for today's affinity-based purification of a GST-tagged protein. FIRST: SECOND: THIRD: FOURTH:

equilibration binding wash elution

Concerning the change in absorbance for a reactant or product over time, the observation of a "plateau" region suggests the reaction has reached _______________.

equilibrium

What does "GST" stand for? Please provide the FULL NAME, no abbreviations. Don't forget to mind your spelling!

glutathione S-transferase

What tag does the pET-41a(+) vector contain?

glutathione-S-transferase tag

The beta-galactosidase enzyme is encoded by the gene ____________, and its mechanism of action can best be described as a "__________" which cleaves beta-1,4 glycosidic linkages between a galactoside monomer and a second organic group. One by-product of this week's enzyme reaction will be ONP, which is responsible for the appearance of a ____________ color that can be measured at ________ nm.

lacz, hydrolase, yellow, 420

Which of the following is used as the target vector in this lab?

pET41-a

Individually speaking...the most important condition/factor that affects an enzyme's activity is typically the __________ of the enzyme's local environment.

pH

What does "PAGE" stand for (three words); please use the full name, no abbreviations. PLEASE MIND YOUR SPELLING!

polyacrylamide gel electrophoresis

Agarose gel electrophoresis separates DNA fragments by:

size only

Structural biochemistry considers the 3D molecular shape of primarily proteins, and how their ________ affects their _________.

structure, function

Prior to running your eluted sample from Lab #9 last week, you will FIRST enzymatically "cleave" the GST-tag from your recombinant fusion protein. The enzyme utilized for this action is called "_______________".

thrombin

The "peak" position on an free energy diagram (represented as the chemical species "AB--C", see image below) following a reaction's progress from reactants/substrates to the products best represents the "____________________" (two words). Please mind your spelling!

transition state

In order to calculate the specific activity of a given enzyme, we must first calculate the enzyme's ___________ under defined conditions. We also need to know the relative ___________ of enzyme used in the assay sample

velocity, quantity