Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

The scope of the research focused on the development and optimization of a protocol for the transformation of chlorella vulgaris. We aim to create a stable transgenic strains of chlorella vulgaris, using agrobacterium tumefaciens and a specific plasmid, pCAMBIA 13 02 that allowed them to insert gene of interest and the potential for biotechnological applications. Metabolic engineering and synthetic biology, microscopy and imaging, CRISPR cost, nine gene editing, bioinformatics, computational modeling, remote sensing, and satellite imaging, nanotechnology bi-processing and downstream processing.

This study introduces an optimized microbacterium tophaceous medium transformation protocol for the microalgae, chlorella vulgaris. Using a specific plasmid and marker. It lays groundwork for enhancing microalgae biotechnology, overcoming previous limitations in reliable transformation tools for C.vulgaris.

Start by diluting 0.5 microliters of the overnight cultures of agrobacterium tumefaciens culture with 50 milliliters of autoclaved ultrapure water. Spread 10 microliters of this diluted culture on lysogeny broth or LB plate. Incubate the plate at 28 to 30 degrees Celsius for one to three days.

Now inoculate a single colony in 20 milliliters of LB medium supplemented with rifampicin. Incubate the culture overnight at 28 to 30 degrees Celsius. The next day, inoculate 500 milliliters of plain LB medium in a shaker flask with nine milliliters of overnight culture.

Place the cultures on a shaker at 28 to 30 degrees Celsius. Following incubation and dividing the cultures into tubes, centrifuge the chilled tubes at four degrees Celsius and 4, 000 G for 15 minutes. Using a pipette, remove the supernatant.

And resuspend each pellet in 50 milliliters of ice cold water. After centrifusion and discarding the supernatant, resuspend the pellet in 25 milliliters of ice cold water in each tube, centrifuge as before. Then resuspend the pellet in one milliliter of ice cold 10%glycerol.

Now combine the contents of all the tubes into a single 50 milliliter tube before centrifuging. Finally, after discarding the supernatent, resuspend the pellet in 400 microliters of ice cold glycerol. Begin by adding 50 microliters of electro competent agrobacterium tumefaciens cells into a pre-chilled 0.1 millimeter cuvette.

Add two microliters of pCAMBIA 13 02 to the cuvette. Now use an electroporater at 2, 400 volts to electroporate the cells. immediately add one milliliter of LB medium to the cuvette and pipette solution up and down to mix gently.

Next, transfer the resuspended cells into a 1.5 milliliter micro tube, then incubate it at 24 to 30 degrees Celsius for a minimum of one hour to recover. Now plate the cells on antibiotic supplemented LB agar. Incubate the plates at 28 to 30 degrees Celsius for two to three days.

Finally, inoculate a single colony in LB broth. Allow the culture to grow overnight. Then cryopreserve the plasmid containing strain with a 50%glycerol solution at minus 80 degrees Celsius for future use.

Begin by pipetting out 0.5 milliliters of a log phase culture of chlorella vulgaris. Spread the culture on a tris acetate phosphate or TAP agar plate. Grow the culture for five days at 25 degrees Celsius.

Next, inoculate 10 milliliters of supplemented LB broth with a loop full of electroporated agrobacterium tumefaciens culture in a shaker flask. Incubate the flask at 28 to 30 degrees Celsius and 250 RPM overnight. The next day, inoculate one milliliter of the overnight culture into 50 milliliters of supplemented LB.Incubate the flask at 28 to 30 degrees Celsius and 250 RPM.

To co-cultivate the algael and bacterial cultures, first transfer the agrobacterium culture into a 50 milliliter tube. Centrifuge the tube at 4, 000 G for 30 minutes at room temperature. Then pipette out the supernatant to discard it and wash the cells twice using the induction medium.

Next, add 25 milliliters of induction medium onto the chlorella vulgaris culture plate. Transfer the cells into a 50 milliliter tube, then centrifuge at 4, 000 G for 15 minutes at room temperature. After disposing of the supernatant, combine the algael cell pellet with 200 microliters of the bacterial suspension.

Shake the combined culture in a rotary incubator at 21 to 25 degrees Celsius. Set at 150 RPM for one hour. Spread 200 microliters of the mixed culture onto induction medium plates, supplemented with 15 millimoles of glucose and incubate the plates in the dark at 21 to 25 degrees Celsius for three days.

After three days, collect the microalgae into a flask using 10 milliliters of TAP medium supplemented with 20 milligrams per liter of tetracycline. Incubate the flask in the dark for two days, maintaining a temperature between 21 to 25 degrees celsius, plate 500 microliters of the culture onto selective medium supplemented with 20 milligrams per liter of tetracycline. Incubate the plates at 21 to 25 degrees Celsius in darkness for two days before shifting them into an illuminated chamber.

Select single colonies from the transformation plate and streak them onto TAP agar plates. To perform colony PCR, start by adding a small volume of the transformant algael cells to 10 microliters of sterile water. Boil the solution at 98 degrees Celsius for 15 minutes.

Similarly process another PCR template for the confirmation of agrobacterium absence in the sample. Run the PCR samples on a DNA agarose gel with a ladder to verify the size of the resulting fragments. Transformed colonies were able to grow on plates containing hygromycin with cefotaxime.

The wild type colonies did not grow on the plates. Colonies resistant up to 70 milligrams per liter of cefotaxime were obtained. Colony PCR amplicon of pCAMBIA 13 02 was absent in the algael samples.

However MGFP5G in amplicon was detected in all three algael samples. Algael cultures that were repeatedly subcultures of cefotaxime show the absence of the virulence protein e2, indicative of plasmid absence. A significant difference in algael growth of the transformants and wild type strains were observed.

Despite lower growth, the transformant had higher fluorescence levels when normalized for cell density.