Team:OUC-China/Description

Description

How do we think of this project？

Algae Outbreak is one of the major marine disaster for marine life, which also threatens social economy and health of human being. The periodically occurring of algae outbreak, the out-break of Enteromorpha, on coastline has been a stubborn local environmental problem in ShanDong province of China.

At the same time, we response the wave of the third worldwide biofuel, which takes alage as core to explore biofuel. Compared with traditional plant used for biofuel like corn, straw and sugarcane, alage has little lignin and more fexible cellulose, so it can be transformed into fuel easier. And we can use the giant advantage that alage won't compete land resources.

Figure 1.1 Enteromorpha outbreak in Qingdao
Figure 1.2 Traditional materials for cellulose fermentation
Figure 1.3 Cellulose in algae Figure 1.4 Cellulose in plant

Figure 2 Sketch of cellulosome

The efficient degradation of cellulose and hemicellulose in algae waste is the key part of biological transformation. In nature, it is cellulose-decomposing microorganisms able to produce cellulose and xylanase that contribute most to cellulose and hemicellulose degradation. Among them, some anaerobion realize this procedure relying on the cellulosome expressed on their surface, which is a kind of scaffold protein complex assembled with cellulose. In this way, various constituents of enzymes can cooperate well with each other, and its proximity effect allows sufficient reaction in the same system, which empower them of efficient degradation ability.

However, the structure of the cellulosome itself is huge, the burden on the target bacteria is enormous, which greatly restricts the final reaction effect.
In order to solve this problem, the enzyme-catalyzed reaction was combined and the metabolic burden was reduced in the mode of S.cerevisiae. We thought of using E. coli as the enzy-matic reaction platform of S.cerevisiae, using E.coli to express a variety of enzymes/proteins to participate in the production of S.cerevisiae.
Such a co-expression platform can lighten the metabolic burden of our yeast and at the same time, ensure the synergistic effect and proximity effect between enzymes. Ultimately, our degradation efficiency get improved.

Figure 3 Sketch of adhesion platform

Figure 4 Comparison of promoter/terminator of CYC1 and MINI system

This year,we use S.cerevisiaeas our chassis creatures.You know that Fungal promoters often span hundreds of base pairs, nearly ten times the amount of bacterial coun-terparts.The promoters are in big size. And the same is true of the terminator. This size limits large-scale synthetic biology efforts in Yeasts. So, synthesis promoters and terminator, especially minimally sized, are critical for advancing fungal synthetic biology.The mini promoters are com-prised of short core elements that are generic and interoperable and 10 bp UAS elements that impart strong, constitutive function. These synthetic pro-moters and terminations offer several advantages over native sequences, including an easily synthe-sized short length, minimal sequence homology to native sequences, and similar or better perfor-mance characteristics than those of commonly used longer one.

So what we do？

Firstly, we produced ethanol from algae waste to realize waste utilization.
Secondly, we constructed an adhesion platform between heterogeneous cells.
Thirdly, we designed and synthesized a set of concise promoters and terminators in yeast called mini system.

Fermentation

This year, we aim to make use of the cellobiose and xylose produced from waste algae and turn them into ethanol as algae wine, to which we add resveratrol to make it healthy and tasty.
See more at our Design page

Adhesion

we novelly designed and achieved a synthetic biology platform for artificial interspecific cooperation. E. coli and S.cerevisiae are engineered to adhere to each other and form multi-cell functional unit. In this co-culture system, E. coli works as surface-display system of S. cerevisiae for realizing diverse applications of Yeast.
See more at our Design page

Mini system

This year, we work on a mini system including standardized promoters and terminators with concise structure in Yeast, providing more potential for large-scale synthetic biology opera-tions.And the mini system can further improve the expression level of allogenic genes in Yeast.
See more at our Design page

Other experiments

InterLab

This year we are very pleased that we have participated in the interlab project. First, we transformed eight plasmids from the Kit Plate into E. coli DH5-alpha cells. And then use the plate reader to measure the data according to the official protocol. Our data will be aggregated with data from other teams around the world in order to obtain the absolute unit for measuring GFP.
See more at our InterLab page

Improve

Improving E. coli promoters is an important work for synthetic biology. By promoters engineering, we can obtain a different transcription process. While the 5'UTR also has such a function that we can get high expression of the PUTR by combining the natural 5’UTR and the promoter. Researchers are able to provide protein translations by combining natural strong 5'UTRs, and we look for examples in the paper and combine to enhance the promoter. We expect to find those 5'UTRs that can have the potential as a generic enhancement module.
See more at our Improve page

What we have done？

★We successfully registered our team for iGEM at March 20^th.
★We meet all deliverables on the Competition Deliverables page.
★We make a detailed description on what is done by ourselves and what supported by others with precise attribution.
★We participated in the Interlab Measurement and submitted our result.
★We submitted 11 new Biobrick Parts designed by ourselves which play a significant role in our project.
★We communicate and collaborate with nine teams, which functions as a crucial support to each other’s project!
★We carefully confirmed that our work is safe and is of no harm to the environment and society and dig deep into the society for inspiration.
★We spread iGEM spirits and promoted the development of synthetic biology in China through popular science brochure, synthetic biology lecture, summer camp, and social media.
★We participate in various kinds of synthetic biology forum such as Conference of China iGEMer Community (CCiC), Synthetic Biology Young Scholar Forum.
★We improved Part BBa_J23108 by adding a 5’UTR sequence and enhanced the expression level of RFP reporter by 1.5 times!
★We built pathway model for both xylose and cellobiose and agent-based models for adhesion platform. Moreover, we defined the ANRC to analyze the simulation results in ABMs.
★We successfully convert Enteromorpha residue into ethanol and our mini system can apply to different chassis and under various experimental conditions!

Reference

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[4]Curran K A, Morse N J, Markham K A, et al. Short Synthetic Terminators for Improved Heterologous Gene Expression in Yeast[J]. Acs Synthetic Biology, 2015, 4(7):824.
[5]Fan, Li Hai, et al. "Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production." Proceedings of the National Academy of Sciences of the United States of America 109.33(2012):13260.
[6]Fan, L. H., et al. "Engineering yeast with bifunctional minicellulosome and cellodextrin pathway for co-utilization of cellulose-mixed sugars." Biotechnology for Biofuels 9.1(2016):137.
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[8]孙萍, 郭丽琼, 黄佳俊,等. 酿酒酵母工程菌生物合成白藜芦醇[J]. 中国食品学报, 2016, 16(3):68-74.
[9]Zhou, S., et al. "Obtaining a panel of cascade promoter-5'-UTR complexes in Escherichia coli." Acs Synthetic Biology 6.6(2017).