Genetic Technology: Question 7

Worked solution

Part (a): Why the insert changes antibiotic resistance

The plasmid used here relies on a technique called insertional inactivation. The tetracycline-resistance gene is a normal, functional gene until the hGH gene is spliced into the middle of its base sequence. This insertion disrupts the reading frame and sequence of the tetracycline-resistance gene, so the gene can no longer be transcribed and translated to produce a working, functional protein. Bacteria that take up this recombinant plasmid are therefore sensitive to tetracycline.

The ampicillin-resistance gene, however, sits at a different location on the plasmid and is untouched by the insertion. It continues to be transcribed and translated normally, so any bacterium carrying this plasmid, recombinant or not, still produces the functional protein needed to inactivate ampicillin, and so remains resistant to ampicillin.

Part (b): Identifying recombinant colonies by replica plating

1. The bacterial culture, after being mixed with the plasmids, is spread onto a master plate of agar containing ampicillin. Only bacteria that have taken up a plasmid of any kind survive and grow into colonies, because all plasmids retain a functional ampicillin-resistance gene.
2. A sterile velvet pad (or similar) is pressed gently onto the surface of this master plate, picking up a sample of cells from every colony, then pressed onto a fresh plate of agar containing tetracycline, transferring cells from each colony to the same relative position as on the master plate.
3. After incubation, the two plates are compared position by position:
   • A colony present at a given position on both plates took up a plasmid that still has a functional tetracycline-resistance gene, i.e. the original plasmid, with no hGH gene inserted.
   • A colony present on the ampicillin master plate but missing at the matching position on the tetracycline plate has a disrupted tetracycline-resistance gene, i.e. it took up the recombinant plasmid carrying the hGH gene.
4. The corresponding colonies can then be picked directly from the original master plate (which has not been exposed to tetracycline) and cultured further to produce hGH.

Part (c): Why plasmid-free bacteria die on ampicillin

Ampicillin resistance in these bacteria depends entirely on a gene carried on the plasmid, not on the bacterial chromosome. A bacterium that did not take up a plasmid at all, recombinant or otherwise, has no copy of this resistance gene and so cannot produce the protein that would normally protect it from ampicillin. Ampicillin therefore continues to disrupt the bacterium’s cell wall synthesis, killing the cell before it can divide and form a visible colony.

Final answers

• (a) The hGH gene disrupts the tetracycline-resistance gene, so recombinant bacteria are tetracycline-sensitive but keep the undisrupted ampicillin-resistance gene, so remain ampicillin-resistant.
• (b) Colonies are grown on ampicillin, transferred by replica plating to a matching position on a tetracycline plate, and any colony that grows on ampicillin but not tetracycline (at the same position) contains the recombinant plasmid with the hGH gene.
• (c) Bacteria with no plasmid have no ampicillin-resistance gene, so ampicillin kills them before they can form a colony.