JoVE Encyclopedia of Experiments
Microbiology
0 views • 4:14 min • October 30th, 2025
Begin with a culture of cyanobacteria.
Centrifuge the culture, then gently wash the pellet to preserve the bacterial surface pili.
Centrifuge again, resuspend the pellet in fresh medium, and transfer to a tube.
Introduce a non-replicating vector with an antibiotic resistance gene. This gene is flanked by DNA sequences that are homologous to regions located upstream and downstream of a target gene in the bacterial genome.
Incubate to allow pili-mediated internalization of the plasmid, which aligns with the homologous sequences and replaces the target gene through recombination, thereby introducing a mutation.
Spread the cells on agar and incubate, allowing the antibiotic resistance gene to express a resistance protein.
Overlay the agar surface with agar containing an antibiotic, and incubate. The resistance protein in mutant cells inactivates the antibiotic, facilitating their survival and colony formation.
Pick the surviving colonies and re-streak on agar containing an antibiotic to segregate the antibiotic-resistant mutants.
After preparing media, cyanobacteria strains, and plasmids, according to the text protocol, set up a fresh culture by inoculating a loop full of cyanobacterial cells
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