8.4.5The Control of Gene Expression (A Level only)

Culture of Transformed Host Cells — AQA A Level Biology 7402 Revision Notes & Topical Questions

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📚 AQA A Level Biology 7402 Hub / Topical Revision

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Lesson summary

8.4.5 Culture of Transformed Host Cells — AQA A Level Biology 7402 notes: Explain how marker genes are used to identify transformed bacteria, and how transformed cells are cultured.After bacteria have been treated with recombinant plasmids, three kinds of cell exist: cells that t…

inactivated
fermenters
Temperature
continuous culture
batch culture
Exam-style question 1

Section 1 of 9The problem: finding transformed cells

After bacteria have been treated with recombinant plasmids, three kinds of cell exist: cells that took up no plasmid, cells that took up a plasmid that closed up without the gene, and cells with the recombinant plasmid.

Only the third kind is wanted, and it is usually a very small proportion of the total.

Marker genes

They are found using marker genes present on the plasmid.

A marker gene is a gene whose product is easy to detect, so that the presence of the plasmid, or of the inserted gene, can be seen.

Section 2 of 9Antibiotic resistance markers

A typical vector carries two genes for antibiotic resistance, for example to ampicillin and tetracycline. The gene of interest is inserted inside one of them, say the tetracycline resistance gene, so that it is inactivated (insertional inactivation).

Bacteria are spread on agar containing ampicillin. Only bacteria with a plasmid (any plasmid) grow, because they carry the ampicillin resistance gene.

Colonies are copied by replica plating onto agar containing tetracycline, keeping the same positions.

Not

Colonies that grow on ampicillin but not on tetracycline contain the recombinant plasmid, because the tetracycline resistance gene has been disrupted.

These colonies are picked from the original ampicillin plate and cultured.

Section 3 of 9Fluorescent and enzyme markers

Antibiotic markers have a drawback

there is concern about spreading resistance genes to other bacteria.
Alternatives are:

GFP

(green fluorescent protein): the gene of interest is inserted into the GFP gene, so transformed colonies do not fluoresce under UV light, while colonies with a plasmid lacking the insert do. Other designs link GFP so that the opposite is seen.

lacZ

lacZ encodes beta-galactosidase, which turns a colourless substrate (X-gal) blue. Insertion of the gene into lacZ gives white colonies for recombinants, while non-recombinant colonies are blue.

Markers allow simple visual selection and rapid screening of thousands of colonies, and fluorescence can even be used to sort cells automatically.

Section 4 of 9Culturing the transformed cells

Selected cells are grown in large fermenters (bioreactors) in sterile conditions. The conditions are controlled to maximise growth and product yield:

Nutrients

Nutrients glucose as an energy source, a nitrogen source, minerals and vitamins.

Temperature

Temperature kept at the optimum for enzymes of the host, using a water jacket, because respiration releases heat.

pH monitored by probes and buffered, because metabolism changes the pH and enzymes are sensitive to it.

Oxygen

Oxygen supplied by sparging air and stirring, for aerobic respiration.

Sterility

Sterility prevents competing microbes using the nutrients or contaminating the product.

In continuous culture nutrients are added and product removed constantly; in batch culture the vessel is emptied and cleaned after each run, so the culture passes through lag, exponential, stationary and death phases.

Section 5 of 9Aseptic technique

When streaking plates in the laboratory, sterilise the loop in a flame, work near a Bunsen flame to create an updraught, minimise opening the dish, tape the lid with only a few pieces of tape so air still enters, and incubate at about 25 °C to reduce the growth of human pathogens.

Aseptic technique

These steps prevent contamination of cultures and harm to the user.

After use, plates are autoclaved before disposal.

Section 6 of 9Why transformation is inefficient and worked interpretation of plate results

Only a tiny fraction of bacteria take up plasmids, and many plasmids re-close without the gene. If the insert is not orientated correctly it may not be expressed. This is why the screening step is essential, and why multiple markers are used.

Suppose 200 colonies grow on the ampicillin plate.

After replica plating, 188 of them also grow on tetracycline, but 12 do not.

The 188 took up a plasmid that closed without the insert, since both resistance genes still work.

The 12 colonies that failed to grow on tetracycline contain recombinant plasmids, so the transformation efficiency of useful cells is 12 out of 200, which is 6 per cent.

Cells with no plasmid at all would have grown on neither plate and so are not counted.

When describing such results, say both what grew and what that shows about each gene, because the marks are for the reasoning.

Remember that the replica must keep the position of each colony, so that a colony missing from the tetracycline plate can be matched to its twin on the original plate and picked from there, since cells on the tetracycline plate would be killed.

Section 7 of 9Exam-style questions

Colonies of bacteria grow on ampicillin agar but not on tetracycline agar. The gene of interest was inserted into the tetracycline resistance gene. Explain what this indicates. [3]3 marks
Explain two conditions that are controlled in a fermenter. [4]4 marks

Section 8 of 9Key terms

Term	Meaning
Marker gene	Gene used to identify cells that took up a plasmid or recombinant plasmid
Insertional inactivation	Gene disrupted by the insertion of a DNA fragment
Replica plating	Copying colonies from one plate to another
GFP	Green fluorescent protein used as a marker
Fermenter	Large vessel for culturing microorganisms under controlled conditions
Aseptic technique	Methods that prevent contamination of cultures

Section 9 of 9Exam technique

Thinking colonies growing on tetracycline contain the recombinant plasmid, when the gene was inserted into the tetracycline gene.

Describing marker genes as the gene of interest: they are only used to identify cells.

Forgetting the purpose of sterile conditionsto avoid competing microorganisms and contamination of the product.

Saying antibiotics make the bacteria transform: they only select the cells with resistance.

Last updated: 18 October 2026