The value of this protocol is that it allows almost any strain of dictyostelium discoideum to be manipulated by molecular genetics, irrespective of whether that strain will grow axenically in liquid medium or not. The other advantage of this technique is that it is fast and simple. Transpose to transfect cells with an exochromosomal plasmid before starting to mix dictyosteliums.
The results can already be validated two days after transfection using microscopy. It is unusual to work with dictyostelium cells and bacteria suspension. Visualization will help to give experimenters an idea how are the different steps of the protocols are performed.
Demonstrating the procedure with me will be Soudabeh Imanikia, a postdoc from my lab. To begin, prepare 1000 milliliters of sorensen buffer working solution with magnesium chloride and calcium chloride according to the text protocol. After this, use a single colony of K aerogenes to inoculate one liter of LB medium.
Allow the bacteria to grow overnight at 37 degrees celsius with shaking. Spin the cells down in two 500 milliliter centrifuge tubes to harvest the cells. Then, wash the bacteria once with 500 milliliters of buffer.
Resuspend the pellet in 20 milliliters of buffer. Use a photometer to check the optical density of the sample and dilute the sample with buffer until the optical density is about 100. After this, prepare the H40 electroporation buffer according to the text protocol.
Grow K aerogenes to confluence an SM medium overnight at room temperature. Add 400 microliters of bacterial suspension to an SM agar plate and spread it evenly. Then, take a sterile loop and inoculate it with dictyostelium cells and spread the cells at one edge of the plate.
Incubate the plate at 22 degrees Celsius for two days, to ensure the growth zones are large enough for transfection. Add 10 milliliters of K aerogene suspension to a 10 centimeter tissue culture-treated Petri dish. Then, use a 10 microliter disposable inoculation loop to scrape cells from the growth zones of the culture plate.
Transfer the cells to a 1.5 milliliter tube, containing ice-cold H40 buffer. Spin the cells for two seconds at 10000 times gravity. Then, discard the supernatant and resuspend the cells in H40 buffer.
Next, add 100 microliters of the cells to a tube containing one to two micrograms of DNA. Pipette up and down to mix and transfer the cell DNA mixture to a pre-chilled electroporation cuvette. Do not add more than two micrograms of DNA.
Higher DNA concentration are toxic for the cells and reduce efficiency. After electroporation, immediately transfer the cells to a prepared 10 centimeter Petri dish and allow the cells to recover for five hours. To select a transfectant for a knock-out, knock-in, or an act five knock-in, detach the cells from the Petri dish by pipetting liquid over the surface.
Then, set up three dilutions of the selective agent according to the text protocol. Finally, distribute 150 microliters of the prepared dilutions into each well of 96-well flat-bottom tissue culture plates. In this protocol, a dual reporter extrachromosomal plasmid system was demonstrated.
32 hours after transfection, the majority of cells expressed both fluorescent labeled fusion proteins. An act five M cherry knock-in and an NC4 was also attempted. Two bands were obtained after running the digest on an agarose gel.
The 4127 base pair band contains the desired construct. The purified DNA was used for the transfection of NC4 cells. Two clones of the NC4 transfection were analyzed via PCR and both showed the predicted band patterns.
Southern blot was then used to validate the results of the PCR. The NC4 grew faster on bacterial lawns than Ax2 cells. After four hours, more of the NC4 cells were able to crawl under the agarose than Ax2 cells.
The NC4 cells were faster and showed stronger chemotactic response to folate than the Ax2 cells. Always use cells from freshly set up SM plates. Never use cells older than four days, otherwise efficiency will drop.
Freshly isolated, nonextenic, white HAP strains are commonly used to address questions concerning a ruching or social behavior of dictyosteliums. Our allows to apply molecular genetics through those isolates.
