Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

This protocol describes how filamentous cyanobacterium that belongs to oscillatoriales can be transformed by natural transformation. It also shows how different kinds of motion can be studied. Natural transformation is the simplest transformation technique, if it works.

Only DNA, cells, and growth medium are required. So far, no other oscillatoriales member could be transformed by natural transformation. We hope that our studies stimulate other groups to try other oscillatoria.

For transformation, make a good, healthy looking-culture and a good DNA preparation. For motion studies, always use an ultrasound-treated culture. Treatment should be fresh.

Demonstrating the procedure will be Nora Weber, a technician from my laboratory. 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.

Cultivate P.lacuna in F2 medium under 50 RPM horizontal agitation in white light for around five days until the estimated optical density at 750 nanometers becomes 0.35. Store the sample at four degrees Celsius. Homogenize the filaments with ultrasound for one minute at maximum power and cycle of one.

Measure the optical density at 750 nanometers and transfer eight milliliters of the medium containing P.lacuna into a six-centimeter Petri dish. Wait for a few minutes until the sample reaches room temperature and then cover the Petri dish with cellophane foil. Place a microscope slide on the X-Y table of a standard microscope with a camera.

Switch on the microscope light and move a 4X or 10X objective into the path of the light. Then, place the Petri dish on top of the slide. Adjust single filaments or filament bundles by the table's X, Y, and Z movements.

Observe the movements of single filaments or bundles and record the movements with a standard microscope camera. Ensure that the objective lens does not touch the liquid. Pipette 0.5 milliliters of a solution containing P.lacuna on the Bacto agar surface of a six-centimeter Petri dish.

Allow the liquid to enter the surface. After around 20 minutes, close the Petri dish and observe the movement of the filaments on the surface using a 4X or 10X objective. Capture the time lapse recordings using an ocular camera and a mini-computer system.

The filaments must first be focused by eye and then through the ocular camera. Ensure that the time interval between the subsequent images is five seconds to one minute. Program the Linux script of the mini-computer to control the time lapse recording.

Prepare LED holders where the five millimeter LEDs are mounted to irradiate an area of 20-millimeters square from below to above. Measure and adjust the LED intensities. Ensure that the whole setting is in a dark room or a closed, dark container.

Place eight milliliters of the medium containing P.lacuna into a six-centimeter Petri dish, close the Petri dish with the lid, and place it on an LED holder so that the LED is in the center of the Petri dish. After typically two days, capture an image of the Petri dish with a smartphone camera aimed directly at the position of the light treatment. Use a holder and a white sheet as a background to ensure the same distance and light conditions for every picture.

Open the ImageJ software, click on File, Open, and select the file. Then, click on Enter. Select the straight button and press the left mouse button to draw a line from one end of the Petri dish to the opposite end.

Ensure that the line passes through the center of the circle of filaments. Click on Analyze and Measure to see the length of the Petri dish. Then, click on Analyze and Plot Profile in the ImageJ menu.

Estimate an average value for the pixel intensity outside the circle and another average value for the pixel intensity inside the circle. Point the mouse on the Y position between these values to estimate the X values of both sides of the circle. Note both values and calculate the difference.

Then select the straight button and draw a line at the mid-maximum value of the pixel intensity. Finally, click on Analyze and then Measure to get the length of the inner cell circle. Integration and segregation of insert after the transformation of P.lacuna with PAK1 is shown here.

A PCR test with outer primers around one week after the transformation typically has two bands on the electrophoresis gel, one with the size of the wild-type band, and one slower migrating band that indicates the insertion of the resistance cassette. Here, lanes 1 to 4 represent PCR products of filaments after seven days, 11 days, 14 days, and 17 days of the isolation of a resistant filament, and lane 5 represents PCR product of wild-type. In the seven-day sample, the insert is present in a small fraction of the chromosomes.

This fraction increases until 17 days, where no wild-type band is visible;that is, the segregation is complete. This image represents the vector pMH1 for sfGHP expression under the control of endogenous phycocyanin beta promoter. P.lacuna homologous sequence, pUC19 vector backbone, and insert with sfGFP and kanamycin resistance are shown here.

In pMH1, the sfGHP gene is placed three prime of the phycocyanin beta gene;therefore, it is driven by the endogenous cpc beta promoter. Fluorescence images of P.lacuna wild-type filaments and after transformation with PAK1, PAK2, PAK3, and pMH1 are shown here. The expression of sfGFP is driven by the cpc 560 promoter, the a2813 promoter, the psbA2S promoter, or the endogenous cpc beta promoter.

The merged images presented here show the motility of Phormidium lacuna. The movement on the agar surface is shown here. The time interval was one minute.

The movement in the liquid medium is presented here. The time interval was 10 seconds. Natural transformation is a simple method.

Just mix plasmid DNA with a resistance cassette in homologous sequences with the cells and let the cells grow on a medium with antibiotics. Genes can be inactivated and proteins can be overexpressed. This is important for any basic research and for biotechnical studies.

In single-cell cyanobacteria where natural transformation is also established, molecular studies on photosynthesis or photoreceptors, et cetera, were addressed.