Difference between revisions of "Team:Georgia State/Culturing"

Introduction to our Algae

Our 3 algae, Symbiodinium microadriaticum, Oxyrrhis marina, and Dunaliella tertiolecta, were obtained from Bigelow Laboratory.

Symbiodinium microadriaticum was grown as our target algae. This is the photosynthetic algae that resides within the coral and is the algae our project strives to modify in order to reintroduce into corals to combat coral bleaching. We aimed to try several different transformation methods in hopes that we can standardize a successful transformation protocol that has not been fully done for this dinoflagellate.

Oxyrrhis marina is a heterotrophic algae that our lab cultivated as a starting point for the initial stages of algal transformation as this dinoflagellate did have a successful transformation referenced in Nuclear gene transformation in a dinoflagellate (Sprecher, Zhang, and Lin 2019). O. marina was viewed as a control in which the transformation within the article was to be replicated. If the transformation results were achieved effectively, we would modify the protocol towards the Symbiodinium. Compared to S. Microadriaticum, O. Marina appears to be more resilient and less particular with its living conditions.

Dunaliella tertiolecta was grown for the sole purpose of sustenance for O. marina. Like Symbiodinium microadriaticum, this algae is photosynthetic and requires sunlight.

How did we culture?

Initially, we began testing out different medias in different volumes to see which media of a specific amount would most effectively sustain and grow the algae. For all three algae, volumes of 10 mL, 25 mL, and 75 mL of each media (ASP-8A, L1 Medium, f/2 Medium, and L1+f/2 Medium) were used to sustain the algae.

• 1 mL of stock Bigelow algae culture was added into 9 mL of media to make the 10 mL cultures.
• 2 mL of stock Bigelow algae culture was added into 25 mL of media to make the 25 mL cultures
• 3 mL of stock Bigelow algae culture was added into 75 m of media to make the 75 mL cultures.

Observations such a movements, patterns, concentration, appearance, and behavior were recorded. Over a course of several weeks, it appeared that the cultures grew more efficiently within the 75 mL flasks. As for the preference of media, S. microadriaticum favored the ASP-8A media out of all the tested medias. O. marina had a preference within the f/2 medium and ASP-8A media. However, a problem arose when they were fed D. tertiolecta within the ASP-8A media as the photosynthetic algae would eventually overgrow alongside its predators making isolation of O. marina become problematic. To add, when the overgrowth of D. tertiolecta was left unattended for a couple weeks, the O. marina were outcompeted and could no longer be seen within its initial flask. For that reason, f/2 medium was chosen to grow the O. marina transformation cultures. Lastly, D. tertiolecta grew substantially within the ASP-8A media out of the four, but because we decided to grow the O. marina in f/2 medium, D tertiolecta was grown within f/2 medium as the avoid mixing media.

Afterwards, a quantitative analyzation of just Oxyrrhis marina and Symbiodinium microadriaticum was conducted to evaluate factors, such as lighting and temperature, that would influence their growth. For these observations, four cultures (S. microadriaticum in ASP-8A media and f/2 medium and O. marina in filtered, autoclaved seawater and f/2 medium) were placed within a hood with regulated lights and another four were placed by the window with solar light. Using a photometer and thermometer, the light intensity and temperature of the two environments were documented alongside the concentrations of each of the four cultures to analyze the growth patterns.

Looking over the data, Symbiodinium grew faster with the regulated lights. While Oxyrrhis grew only slightly better under regulated lights, it seemed to not have much of a preference.