Team:OUC-China/Description

Description

How does our project come into being？

Ethanol from Algae - turning algal bloom into available resources

The outbreak of green algae is a serious natural disaster, which also threatens social economy and the health of human beings. The periodically occurrence of Enteromorpha along the coastline has been a stubborn local environmental problem here in Qingdao, Shandong, China.

At the same time, we respond to the third generation of biofuel production, in which algae, as an ideal source, takes a dominant position. Compared with traditional terrestrial plant used for biofuel like corn, straw and sugarcane, algae has much less lignin and more softer cellulose, which makes it easier to transform into fuel like ethanol.[1] In addition, algae as a marine plant, does not require land source at all. This can be a great advantage in the current world where population makes a big problem and the land is much more precious than ever. Considering these two reasons, our initial project was born. We decided to make use of Enteromorpha residue and turn it into ethanol in a synthetic biology way.

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

Cellulosome - the key for Yeast to utilize cellulose and hemicellulose from Algae

Figure 2 Sketch of cellulosome

It is easy to understand that the efficient degradation of cellulose and hemicellulose in algae residue is the key of this biological transformation. In nature, it is cellulose-decomposing microorganisms able to produce cellulase and xylanase that contribute most to cellulose and hemicellulose degradation. Among them, some kinds of anaerobion perform this complete procedure relying on the cellulosome expressed on their surface, which is a kind of scaffold protein complex assembled with cellulose. In this way, various constituent of enzymes can cooperate well with each other.[2] Its proximal effect and synergistic effect allow sufficient reaction in the shared environment, which empower them of efficient degradation ability.

**Engineered E. coli adhering to Yeast substitutes cellulosome, and improves fermentation efficiency**

However, the structure of the cellulosome itself is complicated, which places a huge burden on yeast and greatly restricts the final react efficiency.[3] And this problem does not only exist here. So it just comes to us that Escherichia. coli might substitute for the function of cellulosome if only adhered to Saccharomyces cerevisiae. Despite the fact that the first few steps of cellulose and hemicellulose degradation have already been a mature procedure in bioindustry thus there is no need for us to start from them and use cellulosome, the adhesion platform can still be applied in many other situations. Such an adhesion platform between heterogeneous cells will certainly lighten the metabolic burden of yeast and hopefully, ensure the synergistic effect and proximal effect when they cooperate with each other. Meanwhile,with E. coli, a model organism, the system has the potential to realize far more functions.[4][5] So here comes a subpart of our project, the adhesion platform.

Figure 3 Sketch of adhesion platform

Minimal Genetic Regulatory Element - A basic research for our project, and also for extensive research and application

Figure 4 Comparison of promoter/terminator of CYC1 and MINI-GRE

Figure 5 The structure of mini promoter

Figure 6 The structure of mini terminator

In the procedure of circuit design, we realize that the large size of basic genetic regulatory parts may contain non-essential sequences, which can be simplified and optimized by synthetic biology work [8]. Therefore, design and synthesis of minimal promoters and terminators are critical for advancing synthetic biology in Yeast SynBio.

In previous research, synthetic biologists have achieved minimal promoters and minimal terminators in yeast. [7-8,13] However, they also find that the combination of promoter and terminator will affect the behavior of the circuit too, which means that it is difficult to predict the output level of particular circuit by the quantitative result of promoter and terminator respectively. Here, we combine the minimal promoter and minimal terminator together as a Minimal Genetic Regulatory Element (MINI-GRE) to test if we could get a minimal promoter-terminator pair with similar or higher transcription output level compared with commonly used transcriptional regulatory elements.

We successfully characterized a minimal promoter-terminator pair (MINI-GRE) with higher output than commonly used one. Simultaneously, we confirmed that this MINI-GRE can provide robust function in different Yeast strains (even in yeast SynX which includes chemical synthetic chromosome) and various environments by iGEM team collaboration. This work set up an important foundation for developing library and toolbox of genetic regulatory elements based on promoter-terminator pair as unit in Yeast, and shows powerful potential to standardize and apply this kind of promoter-terminator pair into various fields.

So what we actually do?

Firstly, we produced ethanol from algae residue to realize waste utilization. Secondly, we constructed an adhesion platform between heterogeneous cells. Thirdly, we designed and synthesized a set of minimal promoter and terminator in yeast called MINI-GRE.

Fermentation

We aim to make use of the cellobiose and xylose produced from waste algae and turn them into ethanol with the help of recombinant S. cerevisiae.[9]

Adhesion

We novelly established a synthetic biological platform for artificial interspecific cooperation. E. coli and S. cerevisiae are engineered to adhere to each other and form a multicellular unit, in which E. coli serves as the surface-display system of S. cerevisiae, having the potential to realize diverse applications of yeast.[6]

MINI-GRE

We work on a MINI-GRE which includes standardized promoters and terminators with minimal structure and even better expression level. Because a simple and concise structure will give convenience to the optimization and engineering of elements, providing more general potential for large-scale synthetic operations in yeast.[7]

See more at our Design page

Other experiments

InterLab

This year we are very pleased to have participated in the interlab project. We transformed eight plasmids from the Kit Plate into E. coli DH5-alpha. And use the plate reader to measure the expression according to provided protocol. Our data will be aggregated with data from other teams around the world to get a better characterization of GFP.
See more at our InterLab page

Improve

Optimization of E. coli promoters is an important work for synthetic biology. Through promoters engineering, we can obtain different transcription levels and dynamic characteristics. Some 5'UTR sequences may affect the function of promoters, so we can improve expression by combining particular 5’UTR and the promoters. Researchers have achieved higher translation level by using natural 5'UTRs on promoters. We searched examples from papers and tried to enhance the promoter by this strategy, expecting 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 met all deliverables on the Competition Deliverables page.
★We made 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 significant roles in our project.
★We communicated and collaborated with nine teams, which functions as a crucial support to each other’s project!
★We carefully confirmed that our work is safe and do not 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 participated in various kinds of synthetic biology forums 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 builted pathway models 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 converted Enteromorpha residue into ethanol and our MINI-GRE can apply to different chassis and under various experimental conditions!

Reference

[1]Jmel, M. A, et al. "Physico-chemical characterization and enzymatic functionalization of Enteromorpha sp. cellulose. " Carbohydrate Polymers 135(2016):274-279.
[2]Artzi, L, E. A. Bayer, and S. Moraïs. "Cellulosomes: bacterial nanomachines for dismantling plant polysaccharides." Nature Reviews Microbiology 15.2(2017):83.
[3]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.
[4]Tanaka T, Masunari S, Ishii J, et al. Displaying non-natural, functional molecules on yeast surfaces via biotin-streptavidin interaction[J]. Journal of Biotechnology, 2010, 145(1):79-83.
[5]Park M, Jose J, Thömmes S, et al. Autodisplay of streptavidin.[J]. Enzyme & Microbial Technology, 2011, 48(4):307-311.
[6]Bloois, Edwin Van, et al. "Decorating microbes: surface display of proteins on Escherichia coli." Trends in Biotechnology 29.2(2011):79.
[7]Redden H,Alper HS,The development and characterization of synthetic minimal yeast promoters[J],Nature Communication,2015,6 : 7810
[8]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.
[9]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.
[10]Sun, Ping, et al. "Combinatorial expression of resveratrol in engineered Saccharomyces cerevisiae." Food & Fermentation Industries 39.8(2013):7-12.
[11]Zhou, S., et al. "Obtaining a panel of cascade promoter-5'-UTR complexes in Escherichia coli." Acs Synthetic Biology 6.6(2017).
[12]Guo, Z. J., and Sherman, F. (1996) Signals sufficient for 3′-end formation of yeast mRNA. Mol. Cell. Biol. 16, 2772−2776."
[13]Yamanishi, M., Katahira, S., Matsuyama, T., 2011. TPS1 terminator increases mRNA and protein yield in a Saccharomyces cerevisiae expression system. Biosci. Biotechnol. Biochem. 75, 2234–2236.