The use of marker genes
Laura Armstrong & Joe Wolfensohn
Teachers
Recall Questions
This topic requires prior knowledge of recombinant DNA. You can test your knowledge on this below.
What is recombinant DNA?
DNA that has been formed by combining DNA from two different organisms.
What is the purpose of a vector in recombinant DNA technology?
To transfer DNA into a host cell (e.g., plasmids or viruses).
What enzyme is used to join DNA fragments into a plasmid?
DNA ligase.
Topic Explainer Video
Check out this @LauraDoesBiology video that explains the use of marker genes or read the full notes below. Once you've gone through the whole note, try out the practice questions!
What Are Marker Genes?
Marker genes are used in recombinant DNA technology to identify cells that have successfully taken up a desired gene (e.g., in a recombinant plasmid).
They allow scientists to screen and select for successful transformations, because not all host cells will take up the new DNA.
Some host cells may not take up a plasmid at all. Some host cells may take up a plasmid but the plasmid may not be recombinant (i.e. not contain the new gene). Some may have taken up a recombinant plasmid with the new gene successfully inserted by DNA ligase - these are the ones the scientists want to culture!
Marker genes allow easy identification of genetically modified cells.
Examples of Marker Genes
1️. Antibiotic Resistance Genes
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Example: Ampicillin resistance and tetracycline resistance genes
The plasmid being used as a vector contains genes for ampicillin resistance and tetracycline resistance (these are the marker genes).
If bacterial cells successfully take up a plasmid, when grown on agar containing ampicillin, they will survive, as they have the gene for ampicillin resistance.
Some of the bacterial cells that survive on the ampicillin agar are then transferred to agar containing tetracycline, in a process known as replica plating.
As the human insulin gene was inserted within the gene for tetracycline resistance, the tetracycline resistance gene has been disrupted. The bacterial cells that successfully took up a recombinant plasmid will not be resistant to tetracycline.
These are the bacterial cells that have been successfully transformed with the human insulin gene.
Even though these cells will be killed on the tetracycline plate, scientists can go back to the original plate (ampicillin agar) to select the bacteria they wish to culture.
2️. Green Fluorescent Protein (GFP) Gene
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A gene from a jellyfish that produces a protein called green fluorescent protein (GFP) can be used as a marker gene. This is inserted into the plasmid.
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The desired gene (such as the gene for human insulin) is transplanted into the centre of the GFP gene.
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This disrupts the GFP gene.
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Bacterial cells that have taken up a plasmid glow when exposed to UV light.
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Bacterial cells that have taken up a recombinant plasmid (with the new gene) will not glow green as the GFP gene has been disrupted. These are the cells scientists will then culture!
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Easier and faster than antibiotic screening - no need to kill cells.
3️. lacZ Gene (β-galactosidase Gene)
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lacZ codes for the enzyme
- galactosidase.
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This lacZ gene can be inserted into a plasmid and used as a marker gene
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In the presence of a special substrate (e.g., X-gal), cells with an active lacZ gene produce a blue colour.
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If the new gene is inserted into the lacZ gene, the lacZ gene is disrupted, and colonies remain colourless.
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Therefore:
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Blue colonies = Cells that have taken up a plasmid (but it did not contain the new gene).
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Colourless colonies = Cells that have taken up a recombinant plasmid (new gene inserted successfully).
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Key Terms
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Marker Gene: A gene used to identify cells that have successfully taken up recombinant DNA.
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Antibiotic Resistance Marker: A gene providing resistance to a specific antibiotic used for selection.
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GFP (Green Fluorescent Protein): A protein that fluoresces under UV light, used as a visual marker.
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lacZ Gene: A gene coding for
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Replica Plating: A method used to transfer colonies to different plates to compare growth patterns.
No answer provided.
Exam Tips
You are not expected to know exactly how all of these different marker genes work. However, make sure you can name examples of marker genes e.g., antibiotic resistance genes, GFP gene.
Make sure you can explain that they are used to determine if a cell has successfully taken up a recombinant plasmid.
No answer provided.
Study the diagrams below.
Results of growth
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Original plate: agar
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Ampicillin plate: contained agar with the antibiotic ampicillin
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Tetracycline plate: contained agar with the antibiotic tetracycline
a) Which bacterial colonies successfully took up plasmids but not containing gene X?
b) Which colonies did take up plasmids with gene X?
a) A, G, H
b) E, F, I
Explained- A, G, H grew on ampicillin so they must have taken up a plasmid (they had the ampicillin resistance gene). However they also grew on tetracycline, indicating the gene for resistance to tetracycline was still intact and functional.
E, F, I grew on ampicillin so they must have taken up a plasmid (they had the ampicillin resistance gene). They were killed by tetracycline, showing the gene for tetracycline resistance had been disrupted by the human insulin gene.
Even though colonies E, F and I were killed on the tetracycline plate, scientists could go back to the ampicillin plate or the original plate to collect these colonies for culture. This is the benefit of using replica plating.
Practice Question
Try to answer the practice question from the TikTok on your own, then watch the video to see how well you did!