Lab 1: molecular cloning, protein purification, and translation

Question 1

What are restriction endonucleases and bacteria plasmids important for?

Answer 1

Modern Gene Development

Question 2

Plasmids

Answer 2

• carry genes for replicating their DNA, transferring themselves from one host to another.
• small, circular, double-stranded DNA molecules found in bacteria, independent of main chromosome.
• they are self-replication and passed to daughter cells during bacterial division.
• used as vectors to insert and propagate specific DNA sequences in host cell.
• want to study a protein, insert its gene into a plasmid and express it in bacteria to produce large amounts of the protein.

Question 3

What two plasmids were used in this experiment?

Answer 3

(1) Mammalian Expression Vectores (e.g., pcDNA3).
- high protein expression (CMV promoter), mRNA stability (BGH signal), and antibiotic resistance for maintenance.

(2) Bacterial expression vectors (e.g., pGEX-GST)
- high protein expression (tac promoter) & tags for protein purification.
- eukaryotic proteins might not express properly due to lack of PTM.

Question 4

Restriction Enzymes

Answer 4

• ‘molecular scissors’ that cut DNA at specific sequences (4-6 nucleotides).
• used in recombinant DNA technology.
• ensure correct DNA orientations in cloning.
• sticky ends - overhanging single strands.
• blunt ends - no overhands.
• use in prokaryotes (defence against foreign DNA (eg., bacteriophages).

Question 5

Ligation Process

Answer 5

• DNA ligase forms phosphodiester bonds to join DNA fragments.
• requires ATP for energy.
• T4 DNA ligase used for both sticky ends and blunt ends.

Question 6

What is the role of Alkaline Phosphatase?

Answer 6

• prevents self-ligation of the vector by removing 5’ phosphates.
• increases the chances of successfully recombinant DNA formation.
• enables virtually unlimited diversity in recombinant DNA molecules.

Question 7

Part 1 of this lab involves molecular cloning of pGEX-GST-4EBP1. What is the goal of this part?

Answer 7

• transfer 4EBP1 DNA fragment from mammalian expression vector (pcDNA3-4EBP1) to a bacterial expression vector (pGEX-GST) to create the recombinant plasmid pGEX-GST-4EBP1.

Question 8

What are the steps in the Restriction Enzyme Digestion?

Answer 8

• 10X restriction enzyme buffer: optimal ionic strength, pH, cofactors (Mg2+) for enzyme activity.
• enzymes BamH1 and XhoI are used to double digest.
• pGEX-GST Vector: linearizes the vector (originally circle, cut to be linear, to prepare vector for insertion of a DNA fragment). It is treated with Calf Intestine Alkaline Phosphatase (CIAP) to remove the 5’ phosphate group and prevent self-ligation.
• pcDNA3-4EBP1 vector: cuts out the 4EBP1 fragment, cut out so can ligate to the pGEX-GST vector and make, pGEX-GST-4EBP1.

Question 9

What is the process in DNA seperation via agarose gel electrophoresis?

Answer 9

• run the digested DNA on an agarose gel to separate fragments.
• pGEX-GST: around 5000 bp.
• pcDNA3-4EBP1: 2 bands
  –> pcDNA3 backbone: around 5387 bp.
  –> 4EBP1 fragment: around 364 bp.

smaller fragments migrate faster
extract the 4EBP1 fragment & pGEX-GST vector from gel.

Question 10

What are the steps in DNA purification?

Answer 10

• use a DNA gel purification kit to isolate the extracted DNA fragments.

Question 11

What is the ligation process?

Answer 11

• Use T4 DNA ligase to join: linearized pGEX-GST vecor with 4EBP1 fragment. This forms the recombinant plasmid.
• L1, L2, L3 represent the 3 ligation conditions.

Question 12

What is the transformation process?

Answer 12

• introduce the recombinant plasmid into E. coli using CaCl2 (males the cells competent for DNA uptake) transformation method.
• render bacteria ‘competent’ to take up foreign DNA.
• mix bacteria with plasmid and plate on agar containing ampicillin.

Question 13

After the transformation process, there is selection of recombinant plasmids. explain this.

Answer 13

• only transformed bacteria with the plasmid grown on ampicillin plates.
• no plasmid: bacteria are ampicillin sensitive and won’t grow.
• original pGEX-GST plasmid: bacteria are ampicillin-resistant due to incomplete digestion or self-ligation. (will grow)
• recombinant pGEX-GST-4EBP1 plasmid: desired bacteria and ampicilin resistant (will grow)

Question 14

How is the verification of cloning done?

Answer 14

• expand selected colonies and purify plasmid using a miniprep kit.
• digest plasmid with PstI to confirm presence presence of 4EBP1. Unique cleavage sites in 4EBP1 and pGEX-GST vector generate specific fragments.
• analyze digestion products on agarose gel stained with Redsafe or ethidium bromide: DNA bands mobility inversly correlates with MW.

Question 15

Protein & Translation Overview

Answer 15

• proteins critical for life, around 44% of human’s dry weight.
• protein synthesis occurs via translation, needs substantial cellular resources.
• translation is highly regulated as it influences cellular metabolism and development.
  -> cis & trans acting factors ensures accurate protein synthesis.

Question 16

Overview of translation initiation

Answer 16

(1) Initial steps:
- mRNA synthesized in nucleus, processed (capping, splicing, polyadenylation) and exported to cytoplasm.
- mature mRNA associates with RNA-binding proteins to form mRNP complex.

(2) eIF’s in translation initiation:
- eIF2 carries tRNAiMet to 40S ribosomal subunit.
- eIF2 needs GTP to bind to TRNAiMet & regulated by eIF2B, which exchanges GDP for GTP.

(3) eIF2 regulation.
- phosphorylation of eIF2 (ser51) inhibits eIF2B activity. halting translation.
- several kinases (e.g., HRI, PKR, mGCN2, PERK) regulate eIF2 phosphorylation, especially under stress.

(4) mRNA recruitment to ribosomes.
- eIF4E binds to 7-me-cap on mRNA, inhibiting recruitment to ribosome.
- under low translation demand, eIF4E is sequestered by 4E-BPs.
- phosphorylation of 4E-BPs releases eIF4F, allowing eIF4E complex with eIF4G & eIF4A.

(5) mRNA structural preparation.
- eIF4A (w/ eIF4B) unwinds mRNA secondary structures.
- eIF3 links 40S ribosomal subunit to eIF4F, facilitating translation.

Question 17

What is the translation experimental overview?

Answer 17

To test the effect of recombinant 4E-BP1 on translation of firefly luciferase mRNA in vitro.

Question 18

Rabbit Reticulocyte Lysate System

Answer 18

• reticulocyte lysates (from enucleated red cells) are used as a non-radioactive system for translation assays.
• translation efficiency is measured by luminescence generated by firefly luciferase activity.
• firefly luciferase activity is directly proportional to translation efficiency.
• assay avoids PTM as luciferase is functional as a monomer.
• experiment provides insight on inhibitor role of 4E-BP1 in translation initiation.

Question 19

In week 1, there is L1, L2, L3. What are the differences between them?

Answer 19

L1: negative control for ligation. only has linearized pGEC-GST without insert (4EPB1) -> C1.

L2: experimental reaction -> C2.

L3: experimental reaction but with more insert to see if increase in insert-ratio-to vector improves ligation efficiency -> C3.

Question 20

Agarose gel 1.5% purpose

Answer 20

Used to separate DNA fragments by size during gel electrophoresis.

Question 21

TBE Buffer

Answer 21

Maintains pH & ionic strength during electrophoresis.

Question 22

Red Safe Stain

Answer 22

• intercalates with DNA to allow visualization under UV light.

Question 23

What are the concepts behind gel electrophoresis?

Answer 23

• DNA negatively charged & migrates toward positive electrode in an electric field.
• smaller fragments migrate faster than larger ones.

Question 24

What are the different buffers used in plasmid isolation?

Answer 24

1) PD1 buffer:
- resuspension buffer
- contains RNase to degrade RNA and tris-HCl to maintain pH.
- helps in resuspending cell pellet.

2) PD2 buffer:
- lysis buffer
- contains SDS (detergent - disrupts cell membrane) and NaOH (alkaline agent - denatures chromosomal DNA & proteins).

3) PD3 buffer:
- neutralization buffer.
- contains potassium acetate to neutralize the alkaline pH.
- precipitates proteins, chromosomal DNA, and SDS, leaving plasmid DNA in supernatant.

4) Wash buffer: contains ethanol to remove salts and contaminants from plasmid DNA.

5) Elution buffer: releases plasmid DNA from silica membrane into a pure form. Often tris-based to stabilize DNA.

Question 25

What is the effect of IPTG induction?

Answer 25

• induces the expression of the recombinant protein by binding to the lax repressor, allowing the transcription of protein from plasmid.

Question 26

How was GST-4EBP1 purified?

Answer 26

• resuspend cell pellet in ice-cold lysis buffer,
• add DDT (reduce disulfide bonds, maintaining protein in functional form) and protease inhibitors (prevent degradation of target proteins by proteases released during cell lysis.

Question 27

Affinity selection

Answer 27

• to purify GST-tagged proteins (FSR-4EBP1) from a cell extract.
• so target protein is purified, free from contaminants, for analysis.

Question 28

What is the effect of SDS sample buffer?

Answer 28

• denature proteins and ensure they are in uniform so can track.

Question 29

How is protein concentration determined?

Answer 29

• using BSA standard curve.
• SDS page analysis.
• confirm expression & purity of GST-4EBP1 & GST proteins.
• check MW of proteins.
• equal amounts of protein loaded.
• see if protein was successfully purified or not.

Question 30

In week 5, translation in vitro was done. Assaying inhibitors of cap-dependent translation. Explain this. what was used as the positive control?

Answer 30

• demonstrate how GST-4EBP1 inhibits translation of capped luciferase mRNA in RRL (by binding to eIF4E).
• positive control: cycloheximide, used, it inhibits protein synthesis by interfering with the translocation step in elongation.