Protein Analysis Module - MurA Purification
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Questions and Answers

What is the role of the 2X-SDS loading buffer in the SDS-PAGE process?

  • To provide nutrients for bacterial growth in the samples.
  • To maintain the pH of the protein samples.
  • To increase the concentration of protein samples.
  • To denature proteins and facilitate their migration through the gel. (correct)

What observation indicates that His(6x)-MurA protein has been successfully eluted from the chromatography column?

  • The increase in imidazole concentration in the flow through sample.
  • The presence of a wide range of protein bands in the elution sample.
  • The absence of any protein bands in the flow through sample.
  • The appearance of a distinct MurA band at approximately 45 kDa in the elution sample. (correct)

In the context of the bacterial growth curve investigation, what factor is being examined in week 1?

  • Effect of protein overexpression on E. coli growth.
  • Effect of gene knockout on E. coli growth at different pHs. (correct)
  • Effect of imidazole concentration on bacterial growth.
  • Effect of temperature on the growth of MurA proteins.

Why is imidazole used in the elution buffer for purifying His(6x)-MurA protein?

<p>It competes with the His(6x) tag for binding to Nickel, allowing elution. (D)</p> Signup and view all the answers

What is the goal of investigating MurA protein overexpression in the context of fosfomycin resistance?

<p>To enhance E. coli growth in the presence of the antibiotic. (A)</p> Signup and view all the answers

What is the primary purpose of the Ni-NTA affinity chromatography in the purification process?

<p>To bind His(6x)-MurA protein by its affinity for nickel (A)</p> Signup and view all the answers

At what optical density (O.D.) should E. coli cells be induced to express His(6x)-MurA protein?

<p>0.4-0.5 (A)</p> Signup and view all the answers

What role does the wash buffer play in the purification process?

<p>It allows unwanted proteins to pass through the column (C)</p> Signup and view all the answers

What is the function of imidazole in the elution step of protein purification?

<p>To compete with His-tag for nickel binding (A)</p> Signup and view all the answers

What is the purpose of using the Bradford assay during the protein purification process?

<p>To determine the concentration of the purified protein (D)</p> Signup and view all the answers

Why is the cell pellet centrifuged and lysed in the protein purification workflow?

<p>To collect the supernatant for analysis (D)</p> Signup and view all the answers

What is the purpose of the BugBuster reagent in the purification protocol?

<p>To break down cell membranes and release protein (C)</p> Signup and view all the answers

After binding His(6x)-MurA protein to the Ni-NTA column, what should be done next in the workflow?

<p>Apply the wash buffer to remove unbound proteins (A)</p> Signup and view all the answers

What defines the lag phase of bacterial growth?

<p>Adjustment to the new environment and synthesis of enzymes (C)</p> Signup and view all the answers

What is the primary method through which E. coli reproduces?

<p>Binary fission (A)</p> Signup and view all the answers

What indicates that the minimum inhibitory concentration (MIC) of an antibiotic has been reached?

<p>Cell growth ceases (C)</p> Signup and view all the answers

In the context of antibiotic resistance, what is the function of beta-lactamase?

<p>Inactivates ampicillin (D)</p> Signup and view all the answers

Which phase of bacterial growth is characterized by nutrient depletion and increased waste accumulation?

<p>Stationary phase (C)</p> Signup and view all the answers

What role does the OD measurement play in assessing bacterial growth?

<p>Indicates concentration of living cells based on turbidity (C)</p> Signup and view all the answers

What outcome is expected when E. coli K12 cells with the amp resistance gene are exposed to high concentrations of ampicillin?

<p>They continue to grow despite the antibiotic (D)</p> Signup and view all the answers

What is the purpose of using varying concentrations of the same drug in an antibiotic dose response assay?

<p>To establish the minimum inhibitory concentration (C)</p> Signup and view all the answers

What is the primary function of buffer B in the elution of MurA protein?

<p>To compete with the His tag for binding (D)</p> Signup and view all the answers

Which of the following describes the purpose of the void volume in size exclusion chromatography?

<p>Volume of space between beads (D)</p> Signup and view all the answers

In size exclusion chromatography, which type of molecules elutes first?

<p>Larger molecules (C)</p> Signup and view all the answers

What is the main principle behind affinity chromatography?

<p>Binding through specific interactions with a resin (D)</p> Signup and view all the answers

Why is bovine serum albumin (BSA) commonly used in the Bradford assay?

<p>It serves as a standard for protein quantification (A)</p> Signup and view all the answers

The Beer-Lambert Law applies to which aspect of a Bradford assay?

<p>The relationship between absorbance and protein concentration (B)</p> Signup and view all the answers

What occurs to the color of the solution in a Bradford assay when protein is present?

<p>It changes from brown to blue (D)</p> Signup and view all the answers

How does vitamin B12 behave in the size exclusion chromatography of hemoglobin and vitamin B12?

<p>It elutes slower than hemoglobin (D)</p> Signup and view all the answers

What is the optimal adjustment to the imidazole concentration in the wash buffer to improve purification outcomes in Ni-NTA chromatography?

<p>Increase the imidazole concentration (D)</p> Signup and view all the answers

If you notice a sudden increase in absorbance at 280 nm during sample application but the peak diminishes, what is the likely reason?

<p>Weak protein binding to the column (C)</p> Signup and view all the answers

What might cause significant amounts of protein to remain bound to a Ni-NTA column even after elution with 300 mM imidazole?

<p>The His-tag of the protein is buried, reducing binding affinity (C)</p> Signup and view all the answers

To enhance the binding efficiency of a His-tagged protein during Ni-NTA chromatography, what modification could be implemented?

<p>Lower the imidazole concentration in the binding buffer (B)</p> Signup and view all the answers

Which action is recommended to ensure the elution of proteins remaining bound to the Ni-NTA column after high imidazole elution?

<p>Incorporate EDTA to chelate Ni ions (C)</p> Signup and view all the answers

What could be a consequence of reducing imidazole concentration in the binding buffer too much during protein purification?

<p>Decreased binding of the target protein to the resin (A)</p> Signup and view all the answers

Why is it important to increase the incubation time during the binding step in Ni-NTA chromatography?

<p>To ensure the target protein binds better to the resin (B)</p> Signup and view all the answers

What is one potential downside of having a high concentration of imidazole in the elution buffer?

<p>It may lead to loss of other desired proteins (C)</p> Signup and view all the answers

What is the effect of increasing imidazole concentration in the wash buffer during Ni-NTA purification?

<p>It helps wash away weakly bound contaminants. (B)</p> Signup and view all the answers

Why might a protein elute at a lower imidazole concentration than anticipated during purification?

<p>The His-tag may have been cleaved. (A)</p> Signup and view all the answers

What might be an experimental approach to verify the cause of lower than expected elution concentration?

<p>Using SDS-PAGE to analyze protein degradation. (C)</p> Signup and view all the answers

What is the primary role of MdtM in E. coli under alkaline conditions?

<p>It helps maintain a stable, acidic cytoplasmic pH. (B)</p> Signup and view all the answers

What was the reason for creating the ΔmdtM knockout strain in E. coli?

<p>To study the effects of pH on growth without mdtM. (A)</p> Signup and view all the answers

What was observed regarding growth differences between wild-type and ΔmdtM strains at alkaline pH levels?

<p>Wild-type strains had better growth than knockout strains at pH ≥ 9.0. (A)</p> Signup and view all the answers

What was the purpose of including the carbenicillin resistance gene in the plasmid along with the mdtM gene?

<p>To track whether the bacterium retained the plasmid. (D)</p> Signup and view all the answers

What was the growth condition used to ensure that the ΔmdtM knockout strain was functioning properly?

<p>Inclusion of kanamycin in the growth medium. (C)</p> Signup and view all the answers

Flashcards

MurA protein purification

The process of isolating and separating the MurA protein from other cellular components to obtain a high purity preparation.

AKTA FPLC

A system used for protein purification based on chromatography techniques. It automatically controls the process.

Size exclusion chromatography

A method to separate molecules based on size. Larger molecules elute faster than smaller ones.

Bradford assay

A method used to quantify the concentration of protein in a solution of unknown concentration.

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Ni-NTA affinity chromatography

A chromatography method that isolates proteins tagged with His(6x) by binding to Nickel ions immobilized onto a resin.

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His(6x) tag

A short amino acid sequence that increases protein affinity toward Nickel ions, used in protein purification.

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IPTG induction

A method to trigger the expression of specific proteins from a plasmid.

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Cell lysis

The process of breaking open cells to release their contents into solution.

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Size Exclusion Chromatography

Separates molecules based on size using beads in a column. Larger molecules elute first, smaller ones later.

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Affinity Chromatography

Purifies protein using a resin bound to a specific molecule that attracts the protein of interest.

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Ion Exchange Chromatography

Separates proteins based on their charge, using charged resin to attract/repel proteins.

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Elution Buffer (MurA)

A buffer used to elute (release) a protein (MurA) from a column that's bound to the column by its His-tag.

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Bradford Assay

A method to quantify protein concentration using a dye that changes color in the presence of protein.

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Beer-Lambert Law

A relationship showing how much light absorbance relates to the concentration of a substances in solution.

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Size Exclusion Limit

The upper limit in size for a resin in size exclusion chromatography; things larger than the exclusion limit will elute quickly in the void volume.

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Mobile Phase

The liquid or gas that moves the sample through the chromatographic column or medium.

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SDS-PAGE

A technique used to separate proteins based on their size.

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Protein concentration

The amount of protein in a sample, often measured using a standard curve.

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Affinity chromatography

A technique to purify specific proteins by exploiting their interaction with a specific ligand in a column..

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Bacterial growth curve

Graph tracking changes in bacterial population size over time.

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Gene knockout

A technique to intentionally disable a specific gene in an organism.

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Bacterial Binary Fission

A type of asexual reproduction in bacteria where a single cell divides into two identical daughter cells.

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Bacterial Growth Phases

Four stages of bacterial growth in a liquid medium: lag, log, stationary, and death.

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Minimum Inhibitory Concentration (MIC)

The lowest concentration of an antibiotic that prevents the growth of a bacterial culture.

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Antibiotic Dose-Response Assay

A method to determine the effectiveness of antibiotics by measuring how the growth of the bacteria changes with different antibiotic concentrations.

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Optical Density (OD)

A measure of the turbidity (cloudiness) of a bacterial suspension, reflecting the concentration of cells.

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Ampicillin Resistance

The ability of a bacterial strain to survive the presence of ampicillin due to the inactivation of the antibiotic.

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Beta-Lactamase

An enzyme that breaks down beta-lactam antibiotics, such as ampicillin, effectively rendering them inactive.

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Bacterial Growth Curve

A graphical representation of bacterial growth over time, showing the lag, log, stationary, and death phases.

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Low Elution Imidazole Concentration

Protein elutes from Ni-NTA column at a lower imidazole concentration than expected.

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Improving Elution Purity (Ni-NTA)

Increase imidazole concentration in wash buffer to remove weakly bound contaminants.

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ΔmdtM Knockout Strain

E. coli strain lacking the mdtM gene, used to study its function.

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MdtM Role in pH Homeostasis

MdtM helps E. coli maintain a stable, low cytoplasmic pH relative to the environment.

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MdtM Knockout Strain Growth

Shows difficulty in growing at high pH without MdtM, compared to wild type.

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Plasmid Recovery Verification

Carbenicillin resistance used to ensure bacteria successfully took in plasmid containing the mdtM gene.

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WT Growth Comparison (pH)

Wild-type E. coli grows better than the knockout strain at pH values ≥ 9.0 in liquid culture.

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Verification of Buffer Conditions

Analyzing the buffer solutions for pH, contaminants to understand protein binding issues.

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Imidazole Concentration in Wash Buffer

Increasing the concentration of imidazole in the wash buffer strengthens its binding to contaminants, removing them before target protein elution.

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Transient 280nm Absorbance Increase

A temporary rise in absorbance at 280nm, followed by a decrease, before elution, indicates weak protein binding to the column.

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Decreasing Binding Buffer Imidazole

Lowering the imidazole concentration in the binding buffer strengthens binding between His-tagged protein and the Ni-NTA resin.

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Protein Remaining Bound (After Elution)

Protein remaining bound even after high imidazole elution likely due to buried His-tag or insufficient imidazole concentration to displace tightly bound protein

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Improving His-tagged Protein Binding

Lower imidazole concentration in buffer enhances binding or increase incubation time allows better protein-resin interaction.

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Flow-Through Protein (MurA)

A significant amount of MurA protein in the flow-through suggests insufficient binding to the Ni-NTA resin.

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Optimize Wash Step

Increasing wash buffer imidazole concentration helps remove contaminants before protein elution.

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Protein Binding to Column

Weak protein binding to the column reduces the protein's 'stickiness', sometimes leading to premature elution.

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Study Notes

Protein Analysis Module

  • PA Week 3 Description: MurA protein purification using AKTA FPLC, size exclusion chromatography, Bradford assay for protein concentration.
  • PA Week 4 Description: SDS-PAGE – Coomassie stained gel, MurA protein purification, Bradford assay.
  • MurA Protein Purification (AKTA FPLC): His(6x)-MurA protein purified using Ni-NTA affinity chromatography. His(6x) tag binds nickel, immobilized on resin. Lysate passes through, His(6x)-MurA binds, unwanted proteins are washed away, imidazole elutes MurA.

Workflow: Mura Protein Expression and Purification

  • 1: Monitor uninduced E. coli cell OD (600nm) to ensure they are in optimal log phase.
  • 2: Induce cells with IPTG to express His(6x)-MurA protein.
  • 3: Harvest induced cells, centrifuge to separate pellet (cells) and supernatant (liquid).
  • 4: Lyse cell pellet using BugBuster, centrifuge to remove cell debris.
  • 5: Apply lysate to Ni-NTA column, wash with equilibration buffer. Filter to remove debris. Equilibrate Ni-NTA resin. Allow protein binding via His-tag/Ni affinity.
  • 6: Elute MurA protein using imidazole gradient. Collect elution fractions.

Size Exclusion Chromatography

  • Separates molecules by size.
  • Stationary phase: resin beads.
  • Mobile phase: buffer.
  • Larger molecules elute first.
  • Smaller molecules enter the beads and elute slowly.

Bradford Assay

  • Beer-Lambert Law: linear relationship between absorbance and concentration, but can break down at high concentrations.
  • Bovine Serum Albumin (BSA) used to create a standard curve for protein quantification.

SDS-PAGE

  • 10 µL lysate + 10 µL 2X-SDS loading buffer added to samples.
  • Visualize wide-range of bands, MurA band at ~45 kDa.
  • Flow-through, wash, and elution samples run through SDS-PAGE to visualize protein presence, confirm MurA presence in relevant fractions.
  • Presence/absence of MurA confirmed by specific bands on gels and Western blots.
  • Different samples visualized under same conditions for comparison.
  • Bands on gels (SDS-PAGE) correspond to protein size, using markers of known size for comparison.

Cell-Based Assay Module

  • CBA Week 1 Description: Growth curve to investigate effects of gene knockout or increasing antibiotic concentration on E. coli cell growth
  • CBA Week 2 Description: Investigate MurA protein overexpression to overcome fosfomycin inhibition.
  • Bacteria Growth Curves: Bacterial growth has 4 phases: lag, log, stationary, death.
  • Antibiotic Dose Response Assay: measure cell growth in varying antibiotic concentrations, determine minimum inhibitory concentration (MIC).

MurA Overexpression Dose Response Assay

  • Determine MIC (minimum inhibitory concentration) for two E. coli strains.
  • E. coli strain 1: wild-type (WT)
  • E. coli strain 2: transformed with pCA24N-His(6x)-murA plasmid Measure growth at increasing concentrations of fosfomycin and ampicillin (inhibitors).

MdtM Paper

  • Purpose Of Kanamycin: Maintain internal pH of bacteria (acidic) in alkaline conditions. Keeps mdtM gene expression controlled.
  • Fosfomycin Resistance In E. coli: MurA overexpression increases resistance, potentially via reduced drug uptake or alternative pathways.
  • High Throughput Screening (HTS) Assay: Grow cells in culture, measure growth at various drug concentrations to determine MIC.

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Protein Analysis Module PDF

Description

Test your knowledge on the purification of MurA protein using various techniques such as AKTA FPLC and SDS-PAGE. This quiz covers steps in protein expression, chromatography methods, and assay techniques like Bradford and Coomassie staining. Perfect for students in biochemistry and molecular biology courses!

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