12 Questions
What is the significance of the tetR gene in the plasmid vectors prepared in step 1?
The tetR gene acts as a reporter gene and has a specific recognition site to one of the restriction endonucleases used in step 2.
What are the two restriction endonucleases used to cut the plasmid samples and insulin subunit genes in step 2?
EcoRI and BamHI
Why were the insulin subunit genes created without introns and with an extra codon coding for methionine?
The insulin subunit genes were produced in a laboratory, and the extra codon coding for methionine was added to facilitate the gene expression.
What is the purpose of adding the plasmids to a solution of E. coli bacteria in step 3?
To allow some of the recombinant plasmids to be taken up by the bacteria.
Why were the bacteria cultures spread and incubated onto agar plates containing the antibiotic ampicillin in step 4?
To determine which bacteria successfully took up plasmids.
What is the role of DNA ligase in the creation of recombinant plasmids in step 2?
DNA ligase is used to reestablish the sugar-phosphate backbone and create two different recombinant plasmids.
What is the purpose of testing the colonies with tetracycline after selecting for ampicillin resistance?
To determine which colonies contain bacteria that took up recombinant plasmids, as opposed to non-recombinant plasmids that are also resistant to ampicillin.
What is the function of the ß-galactosidase enzyme in the lacZ gene?
It converts the colourless compound X-gal into a blue-coloured compound, allowing for the identification of bacteria containing recombinant plasmids.
Why is the insulin subunit protein attached to the ß-galactosidase enzyme in a fusion protein?
To protect the smaller insulin subunit protein from digestive enzymes within E. coli that may destroy it.
What is the purpose of adding cyanogen bromide to the insulin subunit and ß-galactosidase fusion proteins?
To break down the methionine that was added at the start of the insulin gene, allowing for the separation of the insulin subunit from the ß-galactosidase.
What is the result of mixing the two insulin chains together?
The formation of connecting disulphide bonds, which allows for the creation of functional human insulin.
What is the purpose of exponentially reproducing the transformed bacteria before breaking down their membranes?
To produce a large amount of the insulin subunit and ß-galactosidase fusion proteins.
This quiz covers the process of creating recombinant plasmid vectors, including the preparation of plasmid vectors containing antibiotic resistance genes and reporter genes. It also involves the use of restriction endonucleases to cut the plasmid DNA. Test your knowledge of molecular biology techniques and gene manipulation.
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