This study details the transformation of the filamentous cyanobacterium Phormidium lacuna using plasmid DNA. The process involves the internalization of DNA via type IV pili and subsequent selection of transformed bacteria.
Take a culture of Phormidium lacuna, a filamentous cyanobacterium.
Homogenize the culture to disperse the filaments, then centrifuge to pellet the bacteria.
Discard the supernatant and resuspend the pellet in nutrient-rich media.
Add plasmid DNA containing green fluorescent protein and antibiotic-resistance genes onto an antibiotic-supplemented agar plate.
Add the bacterial suspension over the DNA and incubate under light conditions.
The bacteria internalize the DNA using type IV pili. The introduced genes integrate into one of the multiple copies of the bacterial genome.
Redistribute the bacteria onto an antibiotic-supplemented agar plate to select the transformed bacteria.
Under a light microscope, identify healthy, green filaments indicating successful transformation.
Transfer the selected filaments to liquid media with a higher antibiotic concentration to enrich the transformed population.
Again, plate them onto agar with higher antibiotic levels for segregating transformants.
Visualize GFP expression under a fluorescence microscope to confirm successful transformation.
Begin by inoculating 50 milliliters liquid F2 medium into each of the two 250-milliliter flasks with one milliliter of P. lacuna filaments from a running culture. Cultivate in white light under agitation for around five days at 25 degrees Celsius.
After five days, homogenize 100 milliliters of P. lacuna cell suspension at 10,000 RPM for three minutes and measure the optical density at 750 nanometers. Then, centrifuge the cell suspension for 15 minutes at 6,000 times g. Remove the supernatant and suspend the pellet in 800 microliters of the remaining liquid and additional F2+ medium. Take eight F2+ Bacto agar plates containing 120 micrograms per milliliter kanamycin and pipette 10 micrograms of DNA into the middle of each agar plate.
Immediately pipette 100 microliters of the cell suspension on top of the DNA. Keep the agar plate without a lid on the clean bench to allow the excess liquid to evaporate. Close the plate and cultivate it in white light at 25 degrees Celsius for two days.
After two days, distribute the filaments of each agar plate onto several fresh F2+ Bacto agar plates containing 120 micrograms per milliliter kanamycin with an inoculation loop. Cultivate the plates in white light at 25 degrees Celsius and check the cultures regularly under a microscope. After 14 to 28 days, identify the dead brownish filaments and search for transformed filaments under the microscope.
The transformed filaments look healthy and green and are different from the bulk of filaments. Transfer each single transformed filament into 50-milliliter flasks with 10 milliliters of F2+ medium with 250 micrograms per milliliter kanamycin. Cultivate in white light at 25 degrees Celsius on a shaker and observe growth for up to four weeks.
Transfer the filaments back to the agar medium containing 250 micrograms per milliliter kanamycin and wait for the filaments to grow. Then, increase the kanamycin concentration again to speed up the segregation. For GFP expression, observes single filaments with a fluorescence microscope at 40X or 63X magnification and capture a bright-field transmission image and a fluorescence image.
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