Team:Hainan China/Poster

Poster: Hainan_China

Introduction

Hainan_China focused on the concept of enriched nutrition for corals and their symbionts to resist bleaching caused by global climate change. We have developed a nutritional enhancement strategy to strengthen coral/Zooxanthellae against marine environmental stress, such as the elevation in temperature and acidification of seawater. Several nutrients were screened and vitamin B₁₂ was selected as the essential nutrient. VB₁₂ producing strain, Pseudomonas denitrificans was used as cellular chassis and vgb gene from marine microalga Vitreoscilla sp., which is mostly upregulated at low oxygen level, as biological part to minimize oxygen demand for effective VB₁₂ production within the framework of synthetic biology. Coral polyps, Zooxanthellae and probiotic P. denitrificans constructs were grown on the microfluidic coral-in-a-chip for successful proof of our probiotic hopythesis. A mariculture tank was built in Sanya Institute of Coral Ecology, China, to grow our "super corals" with enhanced nutrition for a long term, continuous culture.

About Hainan_China

Team PI
Professor Pengcheng Fu

Associate PI
Meng Hu

Student leaders
Yicheng Lou, Siyu Yang, Yunpeng Zhao

Team members
Xinyue Fan, Weile Su, Shuwen Hou, Junkai Wang, Yumeng Li, Peike Guo,
Zhiyi Wang, Chenyu Zhu, Xiyu Zhang, Boran Wang, Zhongrui Li

Problems

Accelerated anthropogenic CO₂ emission has led to global warming. It in turn caused coral bleaching.
In the past two decades, CO₂ emission has led to global warming. As a result, there is an elevation in sea surface temperatures and acidification of seawater which causes symbiont Zooxanthellae to be repelled from the coral polyps. Without Zooxanthellae, corals would lose their colors, which is called bleaching. If the corals remain bleached for certain period of time, it would lead to eventual death of the corals and the destruction of the marine ecosystem.

Inspiration

In analogy to gut microbiota, coral microbiota are able to nourish corals in the marine environment with poor nutrition to enhance their resistance of bleaching. Gut microbiota can digest certain foods to produces important nutritional molecules (e.g., short-chain fatty acids), facilitates absorption of dietary minerals (e.g., magnesium, calcium and iron) and synthesizes some essential vitamins (e.g., vitamin K and folate) and amino acids (i.e., the building blocks of proteins). In analogy, coral microbiota are able to function similarly to nourish corals in the marine environment with poor nutrition to enhance their resistance of bleaching.

Idea

We selected P. denitrificans for efficient production of vitamin B₁₂, vgb gene as a biological part into the cellular chassis P. denitrificans to minimize oxygen demands.
Traditional vitamin B₁₂ production in industry is carried out mainly with the increase of oxygen supply using higher oxygen pumping speed. This leads to higher energy consumption.
we have introduced the vgb gene as a biological part into P. denitrificans to minimize oxygen demands for VB₁₂ production. vgb gene has been proven in favor of extremely low oxygen environment. When vgb gene is transformed into the cellular chassis by synthetic biology work, the P. denitrificans constructs are able to synthesize vitamin B₁₂ at micro-aerobic (or extremely low oxygen) condition.

Hypothesis and Proof

-Proposed probiotic hypothesis that probiotic bacteria would provide nutrition for healthy and stronger coral reefs

-found the key nutrients or metabolites to ensure the growth of healthy zooxanthellae and prevent the coral bleaching

-finally determined vitamin B₁₂ as essential nutrient.

Synthetic Biology

-create a probiotic bacterium to produce vitamin B₁₂, dissolve phosphorus, release potassium, and fix nitrogen

- vgb gene as biological parts, including vgb gene, vgbpro promoter, using pOJ260, E. coli S17, for conjugal transformation into chassis P. denitrificans.

Coral-in-a-chip

We designed microfluidic chips to prove the success of our genetically engineered probiotics on prevention of coral bleaching.

This is the microfluidic chip for long-term observation of coral polyps and zooxanthellae and its microfluidic system.

The coral tissue and zooxanthellae are cultivated in the chip, and our probiotics are added. Then we observed the chip under the microscope. We found that in the group with probiotics, more zooxanthellae are maintaining a symbiotic relationship with coral tissue. This proves that our probiotics work effectively in the prevention of coral bleaching.

And this is the chip we designed to find the optimal nutrient concentrations for the coral tissue and zooxanthellae. Medium of different nutrient concentrations are added by pump and the fluid is distributed by channels and finally forms concentration gradients in the parallel lines of chambers.

Model

After we finished testing the growth condition and the yields of our genetically engineered Pseudomonas denitrificans from 60-140 hours, we built a model to enhance the practicality of our project in real life.

We used curve fitting tools in MATLAB, a MathWorks® product, to obtain the predictive functions based the data from lab.

We divided the prediction problem into two parts: predicting the yields within 60-140 hours and predicting the yields out of 60-140 hours. The second part is further divided into the prediction within and out of the assumption of a turning point in the yields.

This is the predictive function for prediction of fermentation time within 60-140 hours, or out of 60-140 hours with the assumption of one turning point.

This is the predictive function for fermentation time out of 60-140 hours with the assumption of no turning point.

Future

In the future, we will produce a large amount of marine probiotic bacteria.

To further test our probiotic bacteria, we have built artificial Marine coral cultivation tanks in the laboratory of Hainan University during October, 2020. The staff in the laboratory will observe and measure the growth of coral symbionts in the artificial sea

In August 2020, we made an agreement with Sanya Coral Reef Ecological Research Institute to set up an on-site mariculture tank for the co-cultures of Corals, Zooxanthellae and probiotic Pseudomonas denitrificans in real sea water. In the future, the research institute will continue to assist us in collecting data for our continuous coral symbiosis

If the probiotic bacteria are proved to be helpful, we will spread our bacteria into marine regions where coral reefs grow, then the coral bleaching will vanish. Just like the Greek goddess CHLORIS uses her sticker to sprinkle the magic, where she has touched, the spring would come

Spirit of iGEM

-In-depth collaboration with iGEM teams
-partnership with team St. Andrews

-Education and spread
-publish scientific articles on social media

-online discussions, talks, presentations, and forums with the public
-survey
-Sharing and study
-interview experts
-visit research center and institutes

References and Acknowledgements

References

1 SHI HL, WANG ZJ, WU JQ, GUO MJ,CHU J, ZHUANG YP. 2016, Expression of Vitreosicilla Hemoglobin Gene(vgb) In Pseudomonas denitrificans and the Central Carbon Metabolic Flux Analysis on Vitamin B12 Production[J]. China Biotechnology, 36(9): 21-30.
2 Stark BC, Pagilla KR, Dikshit KL, 2015, Recent applications of Vitreosicilla hemoglobin technology in bioproduct synthesis and bioremediation. Applied Microbiology and Biotechnology ,99 (4) : 1627–1636.
3 https://www.lifescience-market.com/plasmid-c-94/poj260-p-108788.html
4 Kara Rogers; Horizontal gene transfer; Encyclopædia Britannica; August 22, 2019; https://www.britannica.com/science/horizontal-gene-transfer
5 Fang, H., Kang, J., & Zhang, D. 2017. Microbial production of vitamin B₁₂: a review and future perspectives. Microbial Cell Factories, 16:15
6 Koike I. & Hattori A. 1975. Energy yield of denitrification: an estimate from growth yield in continuous cultures of Pseudomonas denitrificans under nitrate-, nitrite-, and nitrous oxide-limited conditions. Microbiology, 88, 11-19
7 Matthews JL, Raina JB, Kahlke T, Madeleine JR, van Oppen JH and Suggett DJ, 2020. Symbiodiniaceae-bacteria interactions: rethinking metabolite exchange in reef-building corals as multi-partner metabolic networks 22(5): 1675-1687

Acknowledgements

We are grateful to
Hainan University (China): Dr. Yang Shu, Yichao Qin, and Yinqiang Wu for experimental design and assistance.
Sanya Coral Ecology Institute (China) for wild coral reef application and long term collaboration.
Academic consultants, Dr. Sun and Dr. Wei for their supports in genetic engineering and microfluidic chip.