Flashcards in Experiments to identify the position of the promoter on a plasmid and the direction of transcription Deck (13):
What type of molecule is a plasmid?
A small, circular molecule of DNA that can be replicated separately from the chromosomal DNA in a cell
Where are plasmids commonly used and what for?
Commonly used in molecular biology for cloning and heterologous protein expression
What can the expression of given polymerases be put under the control of?
The lac operon
What does the lac operon consist of?
The lac promoter
The lac operator
The lac I (repressor protein)
What does the addition of IPTG do when added to the medium of cells containing plasmid?
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
What would a produced polymerase need to have?
A ribosome binding site (RBS) for protein translation
A start methionine for initiation
A 6x histidine tag for protein purification
Briefly define affinity chromatography
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
Affinity chromatography can be very useful when..
Recombinant proteins are expressed with affinity tags
How recombinant His-tagged proteins be purified?
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
What is T7 RNA polymerase (T7 RNAP)?
A DNA-dependent RNA polymerase from T7 bacteriophage
What does T7 RNAP do?
Catalysed the formation of RNA in the 5' to 3' direction
Is the T7 polymerase promoter sequence specific on non-specific for this enzyme?
(The promoter sequences are conserved from positions -17 to +3 relative to the start site of transcription)