Experiments to identify the position of the promoter on a plasmid and the direction of transcription

Question 1

What type of molecule is a plasmid?

Answer 1

A small, circular molecule of DNA that can be replicated separately from the chromosomal DNA in a cell

Question 2

Where are plasmids commonly used and what for?

Answer 2

Commonly used in molecular biology for cloning and heterologous protein expression

Question 3

What can the expression of given polymerases be put under the control of?

Answer 3

The lac operon

Question 4

What does the lac operon consist of?

Answer 4

The lac promoter
The lac operator
The lac I (repressor protein)

Question 5

What does the addition of IPTG do when added to the medium of cells containing plasmid?

Answer 5

It binds to the lac repressor protein and releases the repressor from the lac operator, allowing the transcription of genes in the lac operon by the cell’s RNA polymerase

Question 6

What would a produced polymerase need to have?

Answer 6

A ribosome binding site (RBS) for protein translation
A start methionine for initiation
A 6x histidine tag for protein purification

Question 7

Briefly define affinity chromatography

Answer 7

A powerful method to purify a protein of interest, from other proteins and components in a crude cell lysate, based on its specific binding properties to an immobilised ligand

Question 8

Affinity chromatography can be very useful when..

Answer 8

Recombinant proteins are expressed with affinity tags

Question 9

How recombinant His-tagged proteins be purified?

Answer 9

Affinity chromatography using Ni-NTA agarose
Ni-NTA agarose has immobilised nickel ions that bind the histidine residues in the tag
His-tagged proteins bind with high specificity and affinity and contaminating proteins pass through the resin
After washing, His-tagged proteins are eluted with a buffer containing imidazole

Question 10

What is T7 RNA polymerase (T7 RNAP)?

Answer 10

A DNA-dependent RNA polymerase from T7 bacteriophage

Question 11

What does T7 RNAP do?

Answer 11

Catalysed the formation of RNA in the 5’ to 3’ direction

Question 12

Is the T7 polymerase promoter sequence specific on non-specific for this enzyme?

Answer 12

Very specific

The promoter sequences are conserved from positions -17 to +3 relative to the start site of transcription

Question 13

How is the transcription of the T7 RNAP terminated?

Answer 13

By either an RNA hairpin, known as the transcription terminator or, in vitro, by prior cleavage of the DNA with a restriction enzyme such that the template comes to an end, known as a ‘run-off’ transcription assay