This article describes a method for introducing mutations into cyanobacteria using a non-replicating vector. The process involves the use of antibiotic resistance genes and homologous recombination to facilitate genetic modification.
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 into 30 to 50 milliliters of BG11 medium.
Grow the culture in the light at 30 degrees Celsius for two to three days, to an OD750 equal to 0.2 to 0.6.
Following the incubation, centrifuge one to two milliliters of the culture at 2,300 g's for five minutes. Then, after discarding the supernatant, use BG11 medium to wash the pellets once, gently pipetting to resuspend the cells, as vortexing may result in the loss of pili, which are essential for DNA uptake.
After pelleting the cells again and removing the supernatant, add BG11 medium to a final volume of 100 microliters, and transfer the cells to a 14 milliliter round-bottom tube. Next, add 1 microgram of previously prepared plasmid A to the cells, and gently tap to mix.
Then lay the tubes down horizontally in the incubator at 30 degrees Celsius, and incubate for four to six hours. Following the incubation, spread aliquots of the cell culture plasmid DNA mixture on BG11 agar plates without antibiotics, and incubate at 30 degrees Celsius.
Approximately 24 hours later, prepare a 0.6% agar solution in water containing kanamycin and allow it to cool to 42 degrees Celsius. Then add 2.5 to 3 milliliters to the edges of the culture plates and tilt the plate, so the solution forms an even top agar layer on the surface.
Incubate the plates approximately seven days for colonies to be visible. Then divide a fresh BG11 agar plus kanamycin plate into six sectors and use a blunt-end toothpick to streak out individual colonies.
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