In biopharmaceutical manufacturing, recombinant DNA technology is employed to insert the human gene for antithrombin III (a protein that prevents blood clots) into a bacterial plasmid expression vector to transform Escherichia coli\textit{Escherichia coli}Escherichia coli.
Name the specific class of enzyme used to excise the antithrombin gene from donor DNA and cleave the plasmid vector. Explain why utilising the identical enzyme for both procedures is a prerequisite for successful insertion.
Identify the enzyme responsible for permanently sealing the junctions between the human gene insert and the bacterial plasmid vector, and state the precise chemical bond formed during this process.
Rather than isolating the antithrombin gene directly from genomic DNA (which contains non-coding introns), researchers isolate mature mRNA from human liver cells. Describe how this mRNA is converted into double-stranded DNA suitable for ligation into the plasmid.
To select for host bacteria that have successfully integrated the recombinant plasmid, scientists incorporate a marker gene encoding a red fluorescent protein (mCherry). Explain how this marker gene is utilised to identify and isolate the transformed bacterial colonies.
