Genetic Technology: Question 2

Worked solution

Part (a): Preparing a double-stranded copy of the insulin gene

Insulin mRNA carries the base sequence needed to make the insulin protein, but a single strand of RNA cannot be inserted directly into a plasmid to give a stable, expressible gene. Two enzymes are used in sequence:

1. Reverse transcriptase binds to the insulin mRNA and uses it as a template to synthesise a single strand of DNA that is complementary to the mRNA. This first DNA strand is often called complementary DNA (cDNA).
2. DNA polymerase then uses this single cDNA strand as its template, synthesising a second, complementary DNA strand.

The result is double-stranded cDNA, a stable DNA copy of the insulin gene that can now be cut and inserted into a vector.

Part (b): Forming recombinant DNA

Both the double-stranded insulin gene and the bacterial plasmid are cut using the same restriction endonuclease. Because this enzyme recognises the same specific base sequence wherever it occurs, it produces short, single-stranded, complementary sticky ends on both the gene fragment and the cut plasmid.

Since the sticky ends are complementary, the insulin gene can align with the cut ends of the plasmid and base-pair with them (anneal). This holds the gene loosely in place within the plasmid. DNA ligase then catalyses the formation of covalent phosphodiester bonds in the sugar-phosphate backbone on both strands, sealing the gene permanently into the plasmid. The plasmid, now containing the insulin gene, is a molecule of recombinant DNA.

Part (c): Why a promoter is needed

Having the insulin gene present inside a bacterium is not enough on its own for insulin to be made. A promoter is a specific DNA sequence, positioned immediately before the start of a gene, that RNA polymerase must recognise and bind to before it can begin transcribing that gene into mRNA. The human promoter that normally controls the insulin gene may not be recognised by bacterial RNA polymerase, so a promoter that works in bacteria must be inserted along with the gene. Without a suitable promoter, the insulin gene would never be transcribed (or subsequently translated), and no insulin protein would be produced, even though the gene itself is present in the bacterium’s plasmid.

Part (d): Identifying successful bacteria using a marker gene

Not every bacterium in the mixture takes up a recombinant plasmid, so scientists need a way to identify which colonies have succeeded. A marker gene coding for a fluorescent product, such as green fluorescent protein (GFP), is carried on the same plasmid as the insulin gene. When the colonies are grown and then viewed under ultraviolet light:

• Bacteria that took up the recombinant plasmid also gained the marker gene, so they express the fluorescent protein and their colonies glow (fluoresce) under UV light.
• Bacteria that did not take up the plasmid have no marker gene, so their colonies do not fluoresce.

Only the fluorescing colonies therefore contain the recombinant plasmid, allowing them to be selected for further culture and insulin production.

Final answers

• (a) Reverse transcriptase makes a single cDNA strand from the mRNA template; DNA polymerase then makes the second strand, giving double-stranded cDNA.
• (b) Cutting the gene and the plasmid with the same restriction endonuclease gives complementary sticky ends that anneal together, and DNA ligase seals them with phosphodiester bonds to form recombinant DNA.
• (c) A promoter allows the host’s RNA polymerase to bind and initiate transcription of the inserted gene; without it, the gene is not expressed.
• (d) A marker gene coding for a fluorescent product (e.g. GFP) on the recombinant plasmid makes only bacteria that took up the plasmid fluoresce under UV light, identifying successful colonies.