Lab 1: molecular cloning, protein purification, and translation Flashcards
What are restriction endonucleases and bacteria plasmids important for?
Modern Gene Development
Plasmids
- 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.
What two plasmids were used in this experiment?
(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.
Restriction Enzymes
- ‘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).
Ligation Process
- DNA ligase forms phosphodiester bonds to join DNA fragments.
- requires ATP for energy.
- T4 DNA ligase used for both sticky ends and blunt ends.
What is the role of Alkaline Phosphatase?
- 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.
Part 1 of this lab involves molecular cloning of pGEX-GST-4EBP1. What is the goal of this part?
- 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.
What are the steps in the Restriction Enzyme Digestion?
- 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.
What is the process in DNA seperation via agarose gel electrophoresis?
- 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.
What are the steps in DNA purification?
- use a DNA gel purification kit to isolate the extracted DNA fragments.
What is the ligation process?
- 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.
What is the transformation process?
- 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.
After the transformation process, there is selection of recombinant plasmids. explain this.
- 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)
How is the verification of cloning done?
- 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.
Protein & Translation Overview
- 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.
Overview of translation initiation
(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.
What is the translation experimental overview?
To test the effect of recombinant 4E-BP1 on translation of firefly luciferase mRNA in vitro.
Rabbit Reticulocyte Lysate System
- 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.
In week 1, there is L1, L2, L3. What are the differences between them?
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.
Agarose gel 1.5% purpose
Used to separate DNA fragments by size during gel electrophoresis.
TBE Buffer
Maintains pH & ionic strength during electrophoresis.
Red Safe Stain
- intercalates with DNA to allow visualization under UV light.
What are the concepts behind gel electrophoresis?
- DNA negatively charged & migrates toward positive electrode in an electric field.
- smaller fragments migrate faster than larger ones.
What are the different buffers used in plasmid isolation?
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.
