Team:CGU Taiwan/Demonstrate
Demonstration
We are solving problems
4 billion trees are cut down each year to make paper. To secure natural resources, reprocessed paper was developed. Paper reprocessing industry in Taiwan use about 70% of pulp from recycled paper and 30% of primary pulp. Paper recycling includes an important process called deinking. The key of this process is to detach ink from the fibers. Deinking nowadays uses lots of chemical agents including NaOH, NaSiO3, and Na2PO3 . It causes a great amount of pollution and generates huge amounts of waste water. To reduce the pollution from this process, our target is to develop an enzymatic deinking module. Furthermore, in this process a great amount of paper fiber is lost. To solve this problem, our team came up with an idea to replace manufactory process. The plan of our project is to insert enzymatic sequences into yeast by using genetic engineering and use red light to trigger the enzyme expression. After enzyme was expressed, the medium will be put into our process to see if it has the ability to deink.
This one day meet-up event includes brainstorming activities, project presentation and lecture. For the lecture part, we had invited Dr. Wong Chi Huey, the Distinguished Research Fellow from Genomics Research Center, Academia Silica. He shared about his research on Saccharides synthesis and guide us to explore the possibility and future work of synthetic biology. We appreciate for Dr. Wong’s sharing and coming, the speech did broaden the Taiwan iGEMers’ horizons and help us grow.
Key Achievements
● Build up deinking enzyme construct
● Transform construct into yeast
● Let yeast secret out enzyme into medium
● Put medium into our process and shows the dramatic deinking work
● Build up the Hardware device to fulfill deinking process
● Collaborate with TAS, using biofilm to replace flotation chemicals
Overview
The research purpose of whole project is using red light to induce Saccharomyces cerevisiae to secret out xylanase, glucanase and lipase. Using light can preserve more paper fiber. The light induce system was originally built by the 2012 iGEM TU Munich team. Although optogenetics has benefits in our project, one of the disadvantages is it will decrease the amount of protein expression. However the pros outweigh the cons, that is why we are using this system. Also, we made an improvement on this biobrick to counter this protein amount decreasing challenge.
Furthermore, We put manufactory process into lab and use our material to make reprocessed paper. The step-by-step process of paper recycling includes pulping, deinking, floating and pressing. In our process, we move deinking in the very beginning of our process. Instead of using chemical agent to deink, we spray yeast directly on the paper and use light to start the enzyme secretion.
Our Approach
We hope to make a medium that have a deinking ability to demonstrate. We select three enzymes which is xylanase, lipase and glucanase to work with. We assembled these codons with different selection vectors. With different selection vectors, we can transform our construct into yeast and using selection plate to select the success transformed yeast. These yeasts will grow into big volume and use alpha factor to induce our enzyme.
Experimental Design
We first aimed to build up three construct to do the later transform. The first construct is the light induce system which contain two fusion protein and using constitutive TEF promoter. The first protein is PIF3 domain fuse to Gal4 activation domain, the second protein is PhyB domain fuse to Gal4 DNA binding domain. The system we are using is based on the known red light–induced binding of the plant photoreceptor phytochrome to the protein PIF3 and the reversal of this binding by far-red light. By the switch of red light and far red light we can control our enzymes on and off.
The second construct contains xylanase and glucanase. These two enzymes are both triggered by STE12 promoter and working in different directions. STE12 is a promoter which can start working by alpha factor induction.
The third construct contains STE12 protein and lipase. STE12 protein has a Gal1 promoter in front of it and it will start working by two light fusion protein recruit together. Lipase has a STE12 promoter in front. STE12 system is original in yeast genome and in our construct we have a positive feedback which can start up the STE12 production and later binding with our promoter to start our enzyme secretion.
After constructs build, we put plasmids in yeast cell and using alpha factor to start the enzyme secretion. This medium will be sent into our paper making process and proved it has the ability to deink.
Result
Figure1. Hardware deinking device
Figure2. gray scale testing of experiment paper
Figure 2 is we using different medium to replace chemical agent in deinking process. From left to right is PBS, Non-induced medium, induced medium.
Figure2. gray scale testing of experiment paper
Figure 3 is we using different medium to replace chemical agent in deinking process. This is the paper product after pressing and scanning. From left to right is PBS, Non-induced medium, induced medium.
Proof of concept
To proof our concept we first built up three construct and transform constructs into yeast. After that, we use alpha factor to induce the enzyme secretion. This medium will be sent into our paper making process. We use the manufacturing process but replace chemical agent with our medium. What we see is a dramatic difference from our medium and control. The gray scale of PBS only is 235 and noninduce medium is 237. However, when induced medium put into our process the gray scale turns to 243. Gray scale is from 0-255, as the number goes up it means the whiter the paper is. You can see figure 2, paper which use our enzyme to deink is whiter than other sample.
Future work
This year we didn’t finish our project by using light to trigger our system. Instead, we using alpha factor to start the enzyme secretion. In future, we will finish this section of experiment. Furthermore, we want to develop an eco-friendly ink which fit to our device and make deinking more easily. Nevertheless, we want to grow the seed in public’s mind that saving nature is worth costing. That is why we are doing promotion and education.
