Flashcards in Biotech Final - Lecture 2 Deck (22)
What quantity of recombinant protein can one achieve using E. coli.
mg quantities of protein
The expressed protein is purified (affinity) and used for various biochemical experiments. These include?
1) Activity assays
2) Antibody production
3) 3D structure determination
4) Pharmaceutical purposes
What is the log phase?
The rapidly dividing stage of bacteria.
Why is the log phase important?
If cells are too dense, they will deplete the nutrients critical for growth and stop growing-stationary phase. Thus, in protein expression experiments, these are performed when the cells are in log phase.
What is the stationary phase?
If E. coli cells are too dense they will deplete the nutrients critical for growth and stop growing. They enter a so called stationary phase.
What are the different critical components of a genetically engineered plasmid?
1) Selectable marker
3) Bacterial promoter genes
5) Fusion protein
6) Tag (part of fusion protein)
A selectable marker is often what?
What is an MCS?
Multiple cloning site. This is a short segment of DNA containing multiple restriction sites that are typically unique, occurring only once in a given plasmid. Often used in transgenics.
In our case, what would the bacterial promoter be?
The lac operon system.
What would the repressor be?
What does the lacI gene allow?
Selectable induction. Thus we can choose when to induce expression using the lac operon system. In our case this would be during the log phase of bacterial growth.
What would the fusion protein be in our case?
PTEN with a His6 tag.
What is the purpose of a tag?
Used for affinity purification measures, often does not obstruct protein function or structure.
What is IPTG?
A non metabolizable lactose analogue that is a strong inducer of expression.
How is the lac operon regulated?
It is negatively regulated by the transcriptional repressor LacI. In the presence of lactose, or an analogue, binding with lacI allows for a conformational change that decreases its DNA binding activity, eliminating transcriptional repression.
The lac operon promoter is generally located where, relative to the gene of interest?
What are the disadvantages of the E. coli system?
1) Lack of PTM
2) Lack of protein folding
3) Differential codon usage
What is the solution for lack of PTM in the bacterial model?
In-vitro purified protein.
What is the solution to many higher eukaryotic proteins not being able to properly fold in bacterial cells?
Addition of molecular chaperones to assist in folding.
E. coli has less tRNAs for certain amino acids that are prevalent in higher eukaryotic proteins since their genome is AT rich. How does one circumvent this?
Use genetically modified CODON (+) E. coli cells which express extra tRNAs to improve expression.
What is the overview of E. coli expression of PTEN lab?
1) Quantification of cell numbers
3) Cell lysis by sonication
4) Bradford Assay
5) Both the total cell pellet and supernatant of sonicated sample are kept