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

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.

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

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.