When bacteria are mixed with recombinant plasmids, only a small proportion (often around 1%) take up the plasmid. Some plasmids also close up again without the gene. So it is essential to identify the bacteria that have been transformed with the recombinant plasmid.Marker genes
A marker gene on the plasmid shows whether a cell has taken it up. Common markers are:
• antibiotic resistance genes: only bacteria containing the plasmid can grow on agar containing the antibiotic
• genes for fluorescent proteins such as GFP, which make transformed cells glow under ultraviolet light
• genes for enzymes that produce a colour change with a substrate in the agar.Replica plating with two resistance genes
A plasmid with genes for resistance to ampicillin and tetracycline can distinguish all three types of bacteria. The new gene is inserted into the middle of the tetracycline resistance gene, which inactivates it (insertional inactivation).
2. A sterile velvet pad is pressed onto the plate and then onto a plate containing tetracycline, transferring cells in the same pattern. This is replica plating.
3. Bacteria with the original plasmid grow on both plates. Bacteria with the recombinant plasmid grow on ampicillin but are killed by tetracycline.
4. By comparing the plates, the colonies that are missing on the tetracycline plate are identified and taken from the ampicillin plate.Because of concerns about spreading antibiotic resistance, fluorescent and colour markers are increasingly preferred.
A marker gene on the plasmid shows whether a cell has taken it up. Common markers are:
• antibiotic resistance genes: only bacteria containing the plasmid can grow on agar containing the antibiotic
• genes for fluorescent proteins such as GFP, which make transformed cells glow under ultraviolet light
• genes for enzymes that produce a colour change with a substrate in the agar.Replica plating with two resistance genes
A plasmid with genes for resistance to ampicillin and tetracycline can distinguish all three types of bacteria. The new gene is inserted into the middle of the tetracycline resistance gene, which inactivates it (insertional inactivation).
2. A sterile velvet pad is pressed onto the plate and then onto a plate containing tetracycline, transferring cells in the same pattern. This is replica plating.
3. Bacteria with the original plasmid grow on both plates. Bacteria with the recombinant plasmid grow on ampicillin but are killed by tetracycline.
4. By comparing the plates, the colonies that are missing on the tetracycline plate are identified and taken from the ampicillin plate.Because of concerns about spreading antibiotic resistance, fluorescent and colour markers are increasingly preferred.