Team:ULaVerne Collab/project
PROJECT DESCRIPTION
Background
Global climate change results from the release of various gases, such as carbon dioxide (CO2), methane, nitrous oxide, etc., from natural biochemical processes and human activities. The excess release of greenhouse gases from burning fossil fuels form a blanket around Earth’s atmosphere, allowing for the sun’s rays to enter Earth, but preventing them from being reflected back into space. This results in an increase in solar radiation trapped in our atmosphere. 25% of the greenhouse gases plus 90% of the heat trapped by the greenhouse gases are absorbed into the ocean, increasing ocean temperatures by 1-2% every year (FAO, 2017, Lesser, 1996). Furthermore, increasing ocean temperatures has a direct impact on coral reefs, pushing them out of their very narrow climatic niches such that they become bleached (Bellard et al., 2012).
Coral bleaching is defined as the loss of color of Symbiodinium, or the expulsion of Symbiodinium from coral tissues due to the accumulation of reactive oxygen species (ROS) produced in the photosystems of these organisms (Smith et al., 2005).
In ambient conditions, Symbiodinium take up CO2 and light into its chloroplast where the thylakoid/photosystem II will convert CO2 into an ROS known as superoxide anion radical (O2-) (Weis, 2008). O2- moves out of the thylakoid into the chloroplast, where it will be converted into H2O2 by superoxide dismutase (SOD) and can further be converted into O2 by catalase. O2 is released from Symbiodinium and passed on to the coral reef where photons are taken in and transferred throughout PSII and finally to p680, which then transfers its photons to pheophytin.(Tripathy et al., 2012).
Under stressful conditions, such as prolonged exposure to heat in combination with excess light, the pheophytin molecule cannot transfer its photons fast enough to the plastoquinone to carry out the rest of the photosystem reaction. As such, ROS accumulates within the cell and can lead to detrimental effects, such as reacting with other molecules to form toxic molecules that can ultimately lead to cell death. As previously mentioned, cells have a built in mechanism to combat the production of ROS, SOD, which can convert superoxide anion radicals into hydrogen peroxide.
The loss of Symbiodinium from coral tissue is detrimental because corals rely on their relationship with Symbiodinium for survival and supporting a quarter to a third of the ocean’s population (Fujise et al., 2014). In fact, Symbiodinium are responsible for providing corals with 60–85% of their carbohydrates, lipids, and mycosporine-like amino acids, while corals provide protection for Symbiodinium (Goulet et al., 2017).
The causes of expulsion of Symbiodinium from the coral’s tissue is not well understood however, many scientists believe that the primary causes of coral bleaching results from a combination of elevated temperatures and high light intensity (Baker et al., 2008).
Coral bleaching is defined as the loss of color of Symbiodinium, or the expulsion of Symbiodinium from coral tissues due to the accumulation of reactive oxygen species (ROS) produced in the photosystems of these organisms (Smith et al., 2005).
In ambient conditions, Symbiodinium take up CO2 and light into its chloroplast where the thylakoid/photosystem II will convert CO2 into an ROS known as superoxide anion radical (O2-) (Weis, 2008). O2- moves out of the thylakoid into the chloroplast, where it will be converted into H2O2 by superoxide dismutase (SOD) and can further be converted into O2 by catalase. O2 is released from Symbiodinium and passed on to the coral reef where photons are taken in and transferred throughout PSII and finally to p680, which then transfers its photons to pheophytin.(Tripathy et al., 2012).
Under stressful conditions, such as prolonged exposure to heat in combination with excess light, the pheophytin molecule cannot transfer its photons fast enough to the plastoquinone to carry out the rest of the photosystem reaction. As such, ROS accumulates within the cell and can lead to detrimental effects, such as reacting with other molecules to form toxic molecules that can ultimately lead to cell death. As previously mentioned, cells have a built in mechanism to combat the production of ROS, SOD, which can convert superoxide anion radicals into hydrogen peroxide.
The loss of Symbiodinium from coral tissue is detrimental because corals rely on their relationship with Symbiodinium for survival and supporting a quarter to a third of the ocean’s population (Fujise et al., 2014). In fact, Symbiodinium are responsible for providing corals with 60–85% of their carbohydrates, lipids, and mycosporine-like amino acids, while corals provide protection for Symbiodinium (Goulet et al., 2017).
The causes of expulsion of Symbiodinium from the coral’s tissue is not well understood however, many scientists believe that the primary causes of coral bleaching results from a combination of elevated temperatures and high light intensity (Baker et al., 2008).
Our Project
After having a discussion with Rachel Levin, a PhD student that researched extensively in this field, she guided us to first characterize the function of various SODs (FeSOD from Symbiodinium Clade A, FeSOD from Symbiodinium Clade C, and CuZnSOD) under thermal and light intensity stress conditions in Chlamydomonas. Transformation of Symbiodinium is a current problem, as there are no established methods of transformation, so Chlamydomonas will be utilized as a model organism for our project idea. We will upregulate the amount of the different SODs in the cell and test to see if placing them either in the cytoplasm or the chloroplast will result in a higher reduction of ROS under stressful conditions.
In addition, we mathematically modeled our circuits using Comsol to simulate the reaction of O2- with each of the three different SODs. Three models are based on sending each of the SODs to the chloroplast and three other models are based on sending the SODs to the cytoplasm. This helped in distinguishing whether SOD placement in the chloroplast or cytoplasm will result in a quicker reaction rate with O2-. Additionally, this result also informed us which SOD will have the best reaction rate with O2-.
To test whether Chlamydomonas transformed with our circuits of SODs functions like it should in conditions similar to the ocean, a controlled water tank will be created by our maker team that is designed to contain a temperature and UV light sensor to test how well our circuits are working. The creation of this controlled environment will mimic the environmental conditions for algae on coral reefs that are being experienced now and in the future as various problems arise.
Another part of our project was to create a universal plasmid for Symbiodinium. We extracted putative Symbiodinium promoter and terminator sequences from its genome and we will characterize the parts individually to test if they function the way we believe them to. This will tell us if the putative promoter and terminator from its genome is actually an active promoter and terminator in Symbiodinium.
In addition, we mathematically modeled our circuits using Comsol to simulate the reaction of O2- with each of the three different SODs. Three models are based on sending each of the SODs to the chloroplast and three other models are based on sending the SODs to the cytoplasm. This helped in distinguishing whether SOD placement in the chloroplast or cytoplasm will result in a quicker reaction rate with O2-. Additionally, this result also informed us which SOD will have the best reaction rate with O2-.
To test whether Chlamydomonas transformed with our circuits of SODs functions like it should in conditions similar to the ocean, a controlled water tank will be created by our maker team that is designed to contain a temperature and UV light sensor to test how well our circuits are working. The creation of this controlled environment will mimic the environmental conditions for algae on coral reefs that are being experienced now and in the future as various problems arise.
Another part of our project was to create a universal plasmid for Symbiodinium. We extracted putative Symbiodinium promoter and terminator sequences from its genome and we will characterize the parts individually to test if they function the way we believe them to. This will tell us if the putative promoter and terminator from its genome is actually an active promoter and terminator in Symbiodinium.
Results
Figure 5: Chlamydomonas strain cc-1690 growth curve was tested every 12hrs at it’s dark and light cycles by using a spectrophotometer that was set at 750nm. Chlamydomonas under normal conditions at 25 degree Celcius show constant growth until about 96hrs where it shows cells start to die. Chlamydomonas under stress at 33 degrees Celsius shows constant growth, but begins cell death at a rapid rate at about the 50hrs mark
Figure 6: Data from the surveys the high school students took before and after the presentation. The graph is a representation of the student’s responses to the same questions asked before and after the presentation in a pre and post survey. Question 1 was “How supportive are you on synthetic biology (engineering organisms to do things that they don’t normally do)?” There was a significant difference represented by the p-value of 1.67x10-11. In question two students answered more likely when asked “How likely are you to support/fund for the protection of coral reefs?” after the presentation. The change in opinion was significant as the p-value was 0.0087. Questions 3-5 were respectively questioned “How much of an impact do you think climate change has on coral reefs, marine life and humans in the next 10 years?” Overall, there was an increase in the students’ perception of how impactful climate change was. However, question four did not have a significant in responses represented by the p-value of 0.39. The change in responses from questions three and five were impactful as the p-values were 0.00028 and 0.00027 respectively.
Figure 7: Electrophoresis gel from screening colonies of putative Symbiodinium promoter and terminator. Lane 1 is the 1kb ladder. Lane 2 shows pBR9 vector (5114bp) and the terminator. Lane 4 shows the promoter and pBR9 vector (5114bp).
Figure 8: Electrophoresis gel from screening colonies of FeSOD clade A (606bp), FeSOD clade C (600bp), CuZnSOD (463bp) in pBR9 vector (5114bp) and pBR32 vector (5225bp). No bands were found for CuZnSOD in pBR9 vector and pBR32 vector.
References
Bellard CCA, Bertelsmeier C, Leadley P, Thuiller W, Courchamp F. Impacts of climate change
on the future of biodiversity. Ecology Letters. 2012 [accessed 2017 May
7];15(4):365–377.
Goulet TL, Shirur KP, Ramsby BD, Iglesias-Prieto R. The effects of elevated seawater temperatures on Caribbean gorgonian corals and their algal symbionts, Symbiodinium spp. Plos One. 2017 [accessed 2017 May 7];12(2).
Fujise L, Yamashita H, Suzuki G, Sasaki K, Liao LM, Koike K. Moderate Thermal Stress Causes Active and Immediate Expulsion of Photosynthetically Damaged Zooxanthellae (Symbiodinium) from Corals. PLoS ONE. 2014 [accessed 2017 Oct 31];9(12).
Lesser MP. Oxidative stress causes coral bleaching during exposure to elevated temperatures. Coral Reefs. 1997 [accessed 2017 May 7];16(3):187–192.
Food and Agriculture of the United Nations (FAO), Oceans: our allies against climate change [Internet][Updated 2017 May 10].; [cited 2017 October 31]. Available from http://www.fao.org/zhc/detail-events/en/c/1041817/
Smith DJ, Suggett DJ, Baker NR. Is photoinhibition of zooxanthellae photosynthesis the primary cause of thermal bleaching in corals? Global Change Biology. 2005 [accessed 2017 May 7]; 11(1):1–11.
Tripathy BC, Oelmüller R. Reactive oxygen species generation and signaling in plants. Plant Signaling & Behavior. 2012 Dec 1 [accessed 2017 Nov 1]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3578903/
Weis VM. Cellular mechanisms of Cnidarian bleaching: stress causes the collapse of symbiosis. Journal of Experimental Biology. 2008 [accessed 2017 May 7];211(19):3059–3066.
Goulet TL, Shirur KP, Ramsby BD, Iglesias-Prieto R. The effects of elevated seawater temperatures on Caribbean gorgonian corals and their algal symbionts, Symbiodinium spp. Plos One. 2017 [accessed 2017 May 7];12(2).
Fujise L, Yamashita H, Suzuki G, Sasaki K, Liao LM, Koike K. Moderate Thermal Stress Causes Active and Immediate Expulsion of Photosynthetically Damaged Zooxanthellae (Symbiodinium) from Corals. PLoS ONE. 2014 [accessed 2017 Oct 31];9(12).
Lesser MP. Oxidative stress causes coral bleaching during exposure to elevated temperatures. Coral Reefs. 1997 [accessed 2017 May 7];16(3):187–192.
Food and Agriculture of the United Nations (FAO), Oceans: our allies against climate change [Internet][Updated 2017 May 10].; [cited 2017 October 31]. Available from http://www.fao.org/zhc/detail-events/en/c/1041817/
Smith DJ, Suggett DJ, Baker NR. Is photoinhibition of zooxanthellae photosynthesis the primary cause of thermal bleaching in corals? Global Change Biology. 2005 [accessed 2017 May 7]; 11(1):1–11.
Tripathy BC, Oelmüller R. Reactive oxygen species generation and signaling in plants. Plant Signaling & Behavior. 2012 Dec 1 [accessed 2017 Nov 1]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3578903/
Weis VM. Cellular mechanisms of Cnidarian bleaching: stress causes the collapse of symbiosis. Journal of Experimental Biology. 2008 [accessed 2017 May 7];211(19):3059–3066.
