Team:GA State SW Jiaotong/Poster

Poster: GA_State_SW_Jiaotong



SynBio-dinium
Abstract

Coral bleaching, the loss of necessary algal symbionts for the survival of cnidarian reef organisms, is a disastrous environmental issue that is mainly caused by anthropogenic global warming. Genetically modifying corals’ symbiotic microalgae, Symbiodinium, to better withstand heat stress may combat coral bleaching. We have attempted several transformations, creatively designed an algae house, and built a temperature-ecological amplitude model based on Shelford's Law of Tolerance to find the best conditions for culturing Symbiodinium. In addition, we have successfully designed a recombinant plasmid by inserting a GUS reporter gene and the most appropriate modified heat resistant gene, heat shock factor, into a dinoflagellate-optimized expression DinoIII plasmid. We will perform a biolistics gene gun-mediated transformation. Because the commercial gene gun is so expensive, we will use the 2018 iGEM team Worcester’s design to build our own gene gun. Hopefully, the corals will uptake the modified algae, increasing their resistance to bleaching.

Team Members: Chiara Brust, Yilin Lu, Kennex Lam, Zeshi Wang, Krithika Karunakaran, Chen Yang, Asma Khimani, Ningxin Zhu, Quincy Odinjor, Xiaoyi Feng, Ravin Hargrett, Long Gao, Wanjun Luo, Chengshuo Hou, Diva Yadav, Qianjia Zou, Chen Li, Gao Yuyang, Haoran Li, Guodong Tan, Zhihao Tang, Yuchen Jiang

Advisors: Dr. Matthew Brewer, Dr. Xinhe Huang, Dr. Qiankun Zhu, Dr. Jiayu Zhou, Dr. Liao Hai, Tatenda Tela, Amirah Hurst

Problem & Inspiration
We first learned about the extent of this problem through a Netflix documentary Chasing Corals. Coral reefs support a quarter of all marine organisms[1], and the impact of these ecosystems extends well beyond the reefs themselves. Over 500 million people rely on corals to provide food and resources[2]. Coral reefs support the global economy, worth $375 billion each year[2], by providing economic goods and services. After the heatwave in 2016 and 2017, approximately 50% of the Great Barrier Reef bleached[3]. Reef water temperatures around the world are expected to increase even further by 2℃[4]. Without strict proposals to eliminate fossil fuel consumption, marine heatwaves will become more severe[5]. These obstacles, as well as the documentary, inspired our Georgia State University/Southwest Jiaotong University iGEM team to utilize synthetic biology in an attempt to save the corals.
Introduction & Project Design
Introduction
Corals have a symbiotic relationship with algae. The corals provide shelter while the algae provide carbohydrates from photosynthesis. When environmental stressors such as extreme temperatures are present, the algae are expelled from the corals and what’s left is a nearly dead coral skeleton. This is coral bleaching. No one completely understands the exact mechanism behind bleaching, but the loss of this species can have a devastating impact on the world. To prevent this catastrophe, we want to genetically modify corals' algal symbionts to better resist bleaching.

Project Design
Our synthetic biology solution consists of four steps:

1. Culture the algae

2. Introduce bleaching resistant genes into algae

3. Coral reuptake of modified algae

4. Place into reef system
Culturing
Pre-experimental stage of the experiment
We made a plan and purchased all the equipment we needed for algae culturing. Then, we observed the shape of Symbiodinium under a microscope.

Experimental stage---Phase I
First, we grew the algae in a common media called ASP-8A by the window and under LED light in China and the United States.
Three more groups of algae cultures at 18 °C, 22 °C and 26 °C were set up to study the ecological amplitude properties of Symbiodinium. Artificial seawater with salinity of (30±1) PSU was made by ourselves and sterilized through filtration. An air pump was used to provide oxygen; and the illumination time was set up as 12L: 12D. It finally turned out that our medium was contaminated with fungi and bacteria.

Experimental stage---Phase II
Five groups of algae cultures at 20 °C, 22 °C, 24 °C, 26°C and room temperature were set up using the manual climatic box for culturing. This time, we stopped using the air pump. The medium was sterilized with an autoclave, and all the operations were done on a clean bench.

Algae House
In order to efficiently culture the algae, we constructed a bioreactor described as algae house. It could simulate the natural sunlight with 16h light/8h dark. Moreover, you might observe the current temperature of the house on the electronic display. To ensure adequate airflow, we also installed an air pump into each flask.
Resistance Genes

Due to the pandemic, we could not get into the lab, but we were able to carry out the online research phase of our project. In order to identify bleaching resistant genes that can increase the thermal resistance of Symbiodinium, we performed a genomic analysis on different Symbiodinium clades first. From the transcriptome of Symbiodinium, we discovered genes that were differentially expressed at varying temperatures, and forecasted the function of candidate resistance genes according to a phylogenetic analysis. Together with the results of a heat map and a protein-protein interaction analysis from STRING and KEGG analysis, heat shock proteins and heat shock factors were strongly suggested to play a role in thermal resistance of the algae and might be used for our future genetic engineering.


Left) The gene expression profiles of Symbiodinium under normal and high temperature.
Right) Self-made heat map.

Left) Self-made Phylogenetic tree.
Middle) Protein interaction map from STRING.
Right) KEGG pathway map.
Plasmids & Transformations
1. Dino-GUS
Dinoflagellate-optimized plasmid with a codon-optimized GUS reporter gene that will give us a distinguishable “yes or no” if we've successfully transformed or not.
2. pCB302-GUS
Binary expression plasmid that let's us transform agrobacterium and Symbiodinium using the same construct. This plasmid also has a GUS reporter gene.

Here are the two plasmids:Dino-GUS(left) and pCB302-GUS(right).

Nuclear Transformations
We attempted to transform Symbiodinium using multiple nuclear transformations such as Lonza Nucleofector, Agrobacterium-mediated, glass beads, yeast electroporation, and silicon carbide whiskers. However, after learning that Symbiodinium have permanently condensed nuclear chromosomes, which limits access to target gene sites, we had to alter our plan. We are now trying to target its chloroplasts, which are not condensed by using our own homemade biolistic particle delivery system, or gene gun.

Algae Model

Based on the concentration data, we used a model to perform a one-way anova analysis, and found that the maximum specific growth rate was significantly affected by temperature (P<0.01).
Then, we employed the Shelford tolerance law to establish the temperature response formula of Symbiodinium. The growth curve as well as the temperature ecological amplitude of Symbiodinium were also obtained.
In addition, we applied Matlab software to optimize the response formula, and the Newton iterative algorithm was used to estimate and solve the parameters. Finally, the optimal growth temperature, suitable temperature range, and temperature tolerance range for Symbiodinium were determined.

Result
Gene Gun
Description
Our gene gun contains two main parts— dynamic system and emission system, and it is quite easy to operate. First of all, the screen and macrocarrier are installed on the muzzle, and the cells to be transformed are placed under the gun. Secondly, open the air pump at the far end, adjust to the required air pressure. At the same time, make the “air pump to the solenoid valve” section full of gas. Finally, through the relay, the switch of the solenoid valve is turned on, and the transmission is completed within 0.01s.


Improvement
Instead of CO2 bicycle tire inflator, we chose air pump with high pressure due to its reusable and powerful characteristics in our dynamic system. In the emission system part, we designed a detachable pressing block at the muzzle, which could not only fix the macrocarrier and bullet (and keep them a certain distance) effectively, but also facilitate the disassembly of the macrocarrier and bullet.

Human Practices
We accomplished an educational presentation at the middle school affiliated to Southwest Jiaotong University. More than 100 students participated in the presentation and more than 10 students stayed after to ask us questions about the Symbiodinum and its resistance genes at the end of presentation. Through this presentation, we have further popularized coral bleaching and improved awareness of coral protection.


For academic improvement, we consulted with Dr.Lin, a senior researcher in Symbiodium from Xiamen University. We obtained the current academic progress of coral bleaching and Symbiodinium transformation around the world. Moreover, Dr Lin gave us detailed suggestions for our experimental operations.

We also collaborated with the SCU-China iGEM team from Sichuan University. Both teams shared and exchanged experiences and latest research findings. It was helpful to improve our technical achievements in this competition.


Through the Atl reef club forum, we educated the community about our project and asked for their thoughts, opinions, and understanding of the use of GMOs to solve ecological problems. Through back and forth discussions, we provided this community with a lot of knowledge and allowed the lay public to have a voice in scientific concerns.

Results & Conclusions
Algae Growth Curves
We cultured many groups of Symbiodinium to get the growth curves under artificial light and natural sunlight, also at different temperatures.

Model
Constructed by Matlab, we showed the Temperature-ecological amplitude model for Symbiodinium growth using two different versions of the response relation formula to analyze Symbiodinium growth data.

Resistance Genes research findings
After months of research, our team learned how to screen candidate resistance genes from the public data, especially the transcriptome and genome of Symbiodinium. Based on many kinds of Bioinformatic analysis, the heat shock proteins (HSP) and heat shock factors (HSF) were suggested to play an important role in thermal resistance of Symbiodinium.

Gene Gun
In the future, we will continue to improve the gene gun and so as to transform Symbiodinium efficiently and accurately.

Algae House
Our team constructed a portable algae culturing 'house' that can provide the necessary resources for algae growth, including the light, suitable temperature, and CO2.
Lighting system: the color of the light was set to be white for better efficiency in boosting the algae growth.
Temperature monitor: the temperature information was gathered from each culturing containers and shown on a LED screen for reference. Temperature probes can detect temperature with high precision, and it can run underwater.
Temperature adjusting: a heater was set in each container for adjusting. In order to maintain the best effect of growth temperature, the temperature adjusting system program was coded to ensure at least 5 of 8 containers were higher than 24oC, and lower than 25.5oC.

Future Direction

Culturing: Culture Symbiodinium at different temperatures with varying sources of sunlight to obtain more experimental data.

Perfect: Improve and perfect the gene gun and increase its precision by performing multiple transformations using trial and error

Improve: Improve the success rate of transformations by testing the gene gun and exploring new transformation methods.

Test: Test the practicality of preventing coral bleaching by incorporating resistance genes into the algal symbionts.

Reach out: Contact local governments in charge of reef communities for environmental protection.
References & Acknowledgements

[1] Spalding, M., Grenfell, A. (1997). New estimates of global and regional coral reef areas. Coral Reefs 16, 225–230. https://doi.org/10.1007/s003380050078

[2] Cho, Renee. (2011). Losing Our Coral Reefs. State of the Planet. blogs.ei.columbia.edu/2011/06/13/losing-our-coral-reefs/.

[3] Vaughan, Adam. (2019, April 3). The Great Barrier Reef Is Losing Its Ability to Recover from Bleaching. New Scientist. www.newscientist.com/article/2198318-the-great-barrier-reef-is-losing-its-ability-to- recover-from-bleaching/.

[4] Summary for Policymakers of IPCC Special Report on Global Warming of 1.5°C Approved by Governments. The Intergovernmental Panel on Climate Control. www.ipcc.ch/2018/10/08/summary-for-policymakers-of-ipcc- special-report-on-global-warming-of-1-5c-approved-by-governments/.

[5] Oliver, E.C.J., Donat, M.G., Burrows, M.T. et al. (2018). Longer and more frequent marine heatwaves over the past century. Nat Commun 9, 1324 https://doi.org/10.1038/s41467-018-03732-9