Lab 1 Practice Questions Flashcards
Explain the principles behind restriction enzyme digestion and how it is used in molecular cloning.
- Restriction enzymes are endonucleases that recognize and cut DNA at specific sequences, called restriction sites. These enzymes are naturally found in bacteria as a defence mechanism against viruses.
- Each restriction enzyme has a unique recognition sequence, usually 4-8 base pairs long.
- when a restriction enzyme encounters its recognition sequence, it cleaves the DNA molecule, producing fragments with either blunt ends or sticky ends (overhangs).
- In molecular cloning, restriction enzymes are used to cut both the vector (e.g., a plasmid) and the DNA insert at specific sites, generating compatible ends. This allows the insert to be ligated into the vector, creating a recombinant DNA molecule.
- In Lab 1, the restriction enzymes BamHI and XhoI are used to digest the pGEX-GST vector and the pcDNA3-4EBP1 insert, respectively. Both enzymes create compatible sticky ends, facilitating the ligation of the insert into the vector.
Describe the steps involved in preparing competent bacteria and transforming them with a plasmid.
- competent bacteria are bacterial cells that have been treated to increase their ability to take up exogenous DNA, such as plasmids.
- In lab 1, the protocol involves growing E. coli DH5 cells to a specific density, followed by a treatment with CaCl2 solution. This treatment alters the cell membrane, making it more permeable.
- to then transform them into a plasmid, it it incubated on ice, heat shocked, recovers and then is plated on selective media (LB agar plates containing ampicillin - only bacteria that have taken up the plasmid, which carries the ampicillin resistance gene, will be able to grow on this selective medium)
Why is alkaline phosphatase (AP) used in the cloning process?
AP is an enzyme that removes phosphate groups from the 5’ ends of DNA molecules.
In molecular cloning, AP is used to treat the linearized vector after restriction enzyme digestion. This prevents the vector from self-ligating (re-circularizing) without the insert, as ligation requires the presence of a 5’ phosphate group.
By dephosphorylating the vector, the efficiency of ligation between the insert and the vector is increased.
Explain the principle of affinity chromatography and how it is used to purify GST-tagged proteins.
- Affinity chromatography is a purification technique that separates proteins based on their specific binding interactions with a ligand immobilized on a stationary phase (e.g., column)
- in GST-tagged protein purification, the stationary phase is glutathione-sepharose beads, which have glutathione molecules attached to them.
- GST is a protein tag that can be fused to the protein of interest. The GST tag has a high affinity for glutathione.
- Purification steps:
1) Cell lysate preparation.
2) binding to column.
3) washing.
4) elution.
What is the purpose of dialysis in protein purification?
- dialysis is a technique used to separate molecules based on their size using a semi-permeable membrane.
- in protein purification, dialysis is used to remove unwanted small molecules, such as salts, detergents, and small peptides, from the protein solution. These molecules can interfere with downstream applications or affect protein stability.
- the protein solution is placed in a dialysis bag or cassette made of a semi-permeable membrane. The bag is submerged in a large volume of dialysis buffer. The small molecules diffuse out of the bag through the membrane, while the larger protein molecules are retained.
- for example, in our lab, after eluting the GST-4E-BP1 protein from glutathione-sephrarose beads, the eluate is dialysed against Buffer A to remove the glutathione elution buffer. This ensures that the protein is in a suitable buffer for storage and downstream applications.
Describe the principle behind the Bradford assay for protein quantification.
- The Bradford assay is a colorimetric method for determining protein concentration in a solution.
- It relies on the binding of Coomassie Brilliant Blue G-250 dye to proteins.
- When the dye binds to proteins, it undergoes a shift in its absorption spectrum, resulting in a change in colour from reddish-brown (465 nm) to blue (595 nm).
- The intensity of the blue colour is proportional to the protein concentration in the sample.
1) Prepare the standard curve.
2) Reaction with Bradford reagent.
3) Measure absorbance.
4) Determine the protein concentration.
What is the purpose of SDS-PAGE and how does it work?
- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique to separate proteins based on their molecular weight.
- Denaturation: proteins are treated with sodium dodecyl sulphate (SDS), an anionic detergent, and a reducing agent. SDS disrupts the protein’s native structure, unfolds it into a linear polypeptide chain, and coats it with a negative charge. The reducing agent breaks disulphide bonds within the protein.
- Electrophoresis: The denatured proteins are loaded onto a polyacrylamide gel and subjected to an electric field. The negatively charged proteins migrate towards the positive electrode through the gel matrix.
- Seperation by size: The polyacrylamide gel acts as a molecular sieve, separating the proteins based on their size. Smaller proteins migrate faster through the gel pores than larger proteins.
- Visualization: After electrophoresis, the proteins are visualized by staining with Coomassie blue or other protein dyes.
- Molecular weight determination: A protein ladder, consisting of proteins of known molecular weights, is run alongside the samples. By comparing the migration distance of the unknown proteins to the ladder, their molecular weights can be estimated.
How does the rabbit reticulocyte lysate system work for in vitro translation?
- The rabbit reticulocyte lysate (RRL) system is a cell-free system used for in vitro translation, the process of protein synthesis from mRNA outside of a living cell.
- Reticulocytes: Reticulocytes are immature red blood cells that are highly active in protein synthesis. They are a rich source of ribosomes, tRNAs, amino acids, and translation factors required for protein synthesis.
- Lysate preparation: RRL is prepared by lysing rabbit reticulocytes and removing endogenous mRNA and other components that could interfere with the translation of exogenous mRNA.
- In vitro translation reaction: The RRL system contains all the necessary components for protein synthesis. The exogenous mRNA encoding the protein of interest is added to the lysate, along with amino acids and other essential factors. The reaction is incubated at an optimal temperature, typically 30°C, allowing translation to occur.
—> Advantages:
-Simplicity: The RRL system is relatively simple to use compared to other in vitro translation systems.
- High efficiency: RRL is highly efficient in translating a wide variety of mRNAs.
- Post-translational modifications: Some post-translational modifications can occur in the RRL system, making it more representative of in vivo translation.
Explain how GST-4E-BP1 inhibits translation.
- 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) is a protein that regulates translation initiation, the first step in protein synthesis.
- 4E-BP1 binds to eIF4E (eukaryotic translation initiation factor 4E), a protein that binds to the 5’ cap structure of mRNA. This interaction prevents eIF4E from recruiting the ribosome to the mRNA, inhibiting translation initiation.
- GST-4E-BP1: In Lab 1, a fusion protein consisting of GST and 4E-BP1 is used. The GST tag facilitates purification and does not affect the inhibitory activity of 4E-BP1.
- Mechanism of inhibition: By adding increasing concentrations of GST-4E-BP1 to the in vitro translation reaction, the amount of luciferase protein synthesised should decrease. This is because 4E-BP1 binds to eIF4E, preventing the formation of the translation initiation complex.
How does the luciferase assay work, and what does it measure?
- The luciferase assay is a bioluminescence assay used to measure the activity of the enzyme luciferase.
- Luciferase: Luciferase is an enzyme that catalyses the oxidation of luciferin, a substrate, in the presence of oxygen and ATP. This reaction produces light.
- Measurement of luciferase activity:
- Cell lysis: The cells or in vitro translation reactions containing luciferase are lysed to release the enzyme.
- Addition of luciferase assay reagent: The luciferase assay reagent, containing luciferin and other necessary components, is added to the lysate.
- Measurement of light emission: The light emitted from the reaction is measured using a luminometer.
- Applications: The luciferase assay is commonly used as a reporter assay to measure gene expression, promoter activity, and protein-protein interactions. In Lab 1, it is used to measure the efficiency of translation in the presence or absence of inhibitors.
Analyse the results of a hypothetical experiment where different concentrations of GST-4E-BP1 are added to the in vitro translation system.
In an experiment where increasing concentrations of GST-4E-BP1 are added to the in vitro translation system, you would expect to see a dose-dependent decrease in luciferase activity, measured as relative luminescence units (RLUs).
This observation would support the known function of 4E-BP1 as an inhibitor of cap-dependent translation.
Possible results:
- Control (no GST-4E-BP1): High RLU values, indicating efficient translation of the luciferase mRNA.
- Low GST-4E-BP1 concentration: A slight decrease in RLU values compared to the control, indicating some inhibition of translation.
- Medium GST-4E-BP1 concentration: A more significant decrease in RLU values, indicating stronger inhibition of translation.
- High GST-4E-BP1 concentration: A substantial decrease in RLU values, indicating almost complete inhibition of translation.
Explain the importance of positive and negative controls in both the cloning and protein purification experiments.
- Controls are essential components of any experiment, providing a baseline for comparison and ensuring the validity of the results.
- Positive controls: Positive controls are designed to produce the expected result, confirming that the experimental system is working correctly.
- Negative controls: Negative controls are designed to not produce the expected result, demonstrating that the observed effect is specific to the experimental treatment and not due to other factors.
Cloning:
—> Positive control for transformation: A known plasmid that carries the ampicillin resistance gene is transformed into competent bacteria. This control should show a high number of colonies on the LB+ampicillin plates, confirming that the transformation procedure is working correctly.
—> Negative control for transformation: Competent bacteria are not treated with any plasmid DNA. This control should show no colonies on the LB+ampicillin plates, demonstrating that the antibiotic is effective in preventing the growth of non-transformed bacteria.
Protein purification:
—> Positive control for protein expression: A bacterial strain known to express the target protein is induced and processed in parallel with the experimental samples. This control should show a band of the expected size on the SDS-PAGE gel, confirming that the induction and purification procedures are working correctly.
—> Negative control for protein expression: A bacterial strain that does not express the target protein is induced and processed in parallel with the experimental samples. This control should not show a band corresponding to the target protein on the SDS-PAGE gel, demonstrating that the observed band in the experimental samples is specific to the target protein.
Compare and contrast the use of shRNA and CRISPR/Cas9 for controlling gene expression.
Both shRNA (short hairpin RNA) and CRISPR/Cas9 are techniques used to knockdown or knockout gene expression, but they employ different mechanisms.
(1) shRNA:
- shRNA molecules are short RNA sequences that form a hairpin structure.
- They are introduced into cells, where they are processed into small interfering RNAs (siRNAs).
- siRNAs bind to complementary target mRNA, leading to its degradation and reducing gene expression.
(2)CRISPR/Cas9:
- CRISPR/Cas9 is a bacterial adaptive immune system that has been repurposed for gene editing.
- It consists of a guide RNA (sgRNA) that targets a specific DNA sequence and the Cas9 nuclease that cleaves the DNA at the targeted site.
- The DNA cleavage can lead to gene knockout by introducing insertions or deletions during the repair process.
