Biotech Final - Lecture 2

Question 1

What quantity of recombinant protein can one achieve using E. coli.

Answer 1

mg quantities of protein

Question 2

The expressed protein is purified (affinity) and used for various biochemical experiments. These include?

Answer 2

1) Activity assays
2) Antibody production
3) 3D structure determination
4) Pharmaceutical purposes

Question 3

What is the log phase?

Answer 3

The rapidly dividing stage of bacteria.

Question 4

Why is the log phase important?

Answer 4

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.

Question 5

What is the stationary phase?

Answer 5

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.

Question 6

What are the different critical components of a genetically engineered plasmid?

Answer 6

1) Selectable marker
2) MCS
3) Bacterial promoter genes
4) Repressor
5) Fusion protein
6) Tag (part of fusion protein)

Question 7

A selectable marker is often what?

Answer 7

antibiotic resistance

Question 8

What is an MCS?

Answer 8

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.

Question 9

In our case, what would the bacterial promoter be?

Answer 9

The lac operon system.

Question 10

What would the repressor be?

Answer 10

LacI gene.

Question 11

What does the lacI gene allow?

Answer 11

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.

Question 12

What would the fusion protein be in our case?

Answer 12

PTEN with a His6 tag.

Question 13

What is the purpose of a tag?

Answer 13

Used for affinity purification measures, often does not obstruct protein function or structure.

Question 14

What is IPTG?

Answer 14

A non metabolizable lactose analogue that is a strong inducer of expression.

Question 15

How is the lac operon regulated?

Answer 15

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.

Question 16

The lac operon promoter is generally located where, relative to the gene of interest?

Answer 16

Upstream.

Question 17

What are the disadvantages of the E. coli system?

Answer 17

1) Lack of PTM
2) Lack of protein folding
3) Differential codon usage

Question 18

What is the solution for lack of PTM in the bacterial model?

Answer 18

In-vitro purified protein.

Question 19

What is the solution to many higher eukaryotic proteins not being able to properly fold in bacterial cells?

Answer 19

Addition of molecular chaperones to assist in folding.

Question 20

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?

Answer 20

Use genetically modified CODON (+) E. coli cells which express extra tRNAs to improve expression.

Question 21

What is the overview of E. coli expression of PTEN lab?

Answer 21

1) Quantification of cell numbers
- A OD600
2) Centrifugation
3) Cell lysis by sonication
4) Bradford Assay
5) Both the total cell pellet and supernatant of sonicated sample are kept

Question 22

What is the purpose of keeping the supernatant of the sonicated samples?

Answer 22

Lets you know the relative efficiency of sonication.