Protein Analysis Module

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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?

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

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

To enhance E. coli growth in the presence of the antibiotic. Signup and view all the answers

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

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

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

0.4-0.5 Signup and view all the answers

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

It allows unwanted proteins to pass through the column Signup and view all the answers

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

To compete with His-tag for nickel binding Signup and view all the answers

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

To determine the concentration of the purified protein Signup and view all the answers

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

To collect the supernatant for analysis Signup and view all the answers

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

To break down cell membranes and release protein 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?

Apply the wash buffer to remove unbound proteins Signup and view all the answers

What defines the lag phase of bacterial growth?

Adjustment to the new environment and synthesis of enzymes Signup and view all the answers

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

Binary fission Signup and view all the answers

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

Cell growth ceases Signup and view all the answers

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

Inactivates ampicillin Signup and view all the answers

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

Stationary phase Signup and view all the answers

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

Indicates concentration of living cells based on turbidity 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?

They continue to grow despite the antibiotic Signup and view all the answers

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

To establish the minimum inhibitory concentration Signup and view all the answers

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

To compete with the His tag for binding Signup and view all the answers

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

Volume of space between beads Signup and view all the answers

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

Larger molecules Signup and view all the answers

What is the main principle behind affinity chromatography?

Binding through specific interactions with a resin Signup and view all the answers

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

It serves as a standard for protein quantification Signup and view all the answers

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

The relationship between absorbance and protein concentration Signup and view all the answers

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

It changes from brown to blue Signup and view all the answers

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

It elutes slower than hemoglobin 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?

Increase the imidazole concentration 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?

Weak protein binding to the column 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?

The His-tag of the protein is buried, reducing binding affinity 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?

Lower the imidazole concentration in the binding buffer 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?

Incorporate EDTA to chelate Ni ions Signup and view all the answers

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

Decreased binding of the target protein to the resin Signup and view all the answers

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

To ensure the target protein binds better to the resin Signup and view all the answers

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

It may lead to loss of other desired proteins Signup and view all the answers

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

It helps wash away weakly bound contaminants. Signup and view all the answers

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

The His-tag may have been cleaved. Signup and view all the answers

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

Using SDS-PAGE to analyze protein degradation. Signup and view all the answers

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

It helps maintain a stable, acidic cytoplasmic pH. Signup and view all the answers

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

To study the effects of pH on growth without mdtM. Signup and view all the answers

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

Wild-type strains had better growth than knockout strains at pH ≥ 9.0. Signup and view all the answers

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

To track whether the bacterium retained the plasmid. Signup and view all the answers

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

Inclusion of kanamycin in the growth medium. Signup and view all the answers

Study Notes

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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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!

Protein Analysis Module - MurA Purification

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