Team:BUCT/Poster

Poster: BUCT

Legolas•Microcystin:An innovation treatment of algal blooms and microcystin.

Presented by BUCT 2020

PI: Zhengjun Li / Xinxiao Sun

Advisors: Xiaoqi Niu

Student Members: Xuanhe Fan, Haosheng Liu, Huiwen Zheng, Yiqi Zhao, Ao Wang, Yuting Sun, Yixin Bai, Jiayi Gu, Yanjiao Jiang, Jie Zhang, Shuqing Yan, Zhenyao Zhang, JinHong Wang, Zhangzheng You, Zhixing Zhang.

Abstract

Every summer, many lakes around the world are covered with some disgusting and green microbes. They are cyanobacteria, which can produce cyanobacteria toxins, such as microcystins. It can cause brain fever, skin allergies; even induce tumor genesis and liver cancer. In this project, we used a special chassis -cyanophage. We tried to add two parts to the phage: a functional part that can degrade the toxin and a control part that prevents the release of the engineered cyanophage. Our functional part consists of mlrA gene, which expresses microcystin-LR degrading enzyme to decompose the long-lasting cyclic peptide into harmless amino acids. Our control part consists of unnatural amino acid systems. It can be used to limit the proliferation of the cyanophage. By putting our designed recombinant cyanophage into water bodies, we hope this will be a more safe and effective treatment in algal blooms and toxins degradation.

Problem

Cyanobacteria outbreaks have become a topic of conversation in recent years.If cyanobacteria float on the surface, they prevent other creatures from using the sun's rays and living.This seriously disrupts the balance of the waters.The biggest hazard is the production of MC toxins，which can enter and accumulate in the body through drinking water, diet and recreational water, posing a serious threat to the safety of drinking water and human health.

Goals

• Our team has come up with creative ways to use recombinant cyanophages to solve the problem of harmful cyanobacteria.
• We found that mlr gene cluster from Sphingomonas sp. can degrade Microcystin-LR and then reduce its toxicity. We expect to construct recombinant cyanophage carrying the microcystin degrading enzyme to address the concern of algal blooms.
• Safety issues always come first. The codon expansion technology would be applied to ensure the biosafety of recombinant cyanophage.

Dry lab

We refer to the Cellular Automata model to simulate the process of cyanophages acting on cyanobacteria cells, and predict and qualitatively analyze the influence of cyanobacteria reproduction and survival ability on the results.

RGBColor[“#00e2ff”], the blue parts show the solution & the cells that are broken down.
RGBColor[“#339933”], the green parts represent cyanobacterial cells.
RGBColor[“#ffff66”], the yellow parts are the random distribution of phages.
The final result of the phage action shown in the figure is mainly determined by two parameters. By adjusting the spread chance & continue function chance of the phage, we get the system state under different initial parameters and the same action time.

Spread chance=0.1, Continue function chance=0.1

Spread chance=0.1, Continue function chance=0.5

Spread chance=0.3, Continue function chance=0.2

Spread chance=0.4, Continue function chance=0.1

Wet lab

Degradation
We selected the mlrA gene from Novosphingobium sp. THN1 (BBa_K3699001) and BBa_K1378001 from 2014_Peking, adding the promoter J23119, RBS (B0034), and terminator. After that, the entire sequences (BBa_K3699005 and BBa_K3699008) were introduced into pKMV and yielded the recombinant plasmid pKMV-mlrA-THN1 and pKMV-mlrA-C!, which were transformed into E. coli JM109.



Degradation result of pKMV-mlrA-C1 cell extract.

Degradation result of pKMV-mlrA-THN1 cell extract.

We have characterized our MlrA enzyme from Novosphingobium sp. THN1. It is indicated that it can degrade MC-LR with favorable efficiently.
Codon Expansion Technology After the artificial "upgrade" of the cyanophage, we also expect to add "handcuffs" to ensure that the cyanophage would always be implemented in the right place.
The handcuff mentioned above is the “tail” protein with UAG inserted,and the "key" is the codon expansion technology.
We finally decided to use the orthogonal translation system from Methanosarcina mazei. It has been used in E. coli. The J23119 promoter was employed to drive MmBocRS expression. PyltRNA (GenBank: NZ_ Cp009514.1 3998599-3998670) was initiated by proK promoter.


The “tail” protein encodes the putative phage tail sheath protein of Microcystis virus Ma-LMM01. (Protein ID: BAF36182.1)
We have made a base substitution at position 747 (T747G), which converted the Tyrosine codon to a stop codon and lead to the early termination of protein translation.



The introduction of stop codon caused the early termination of protein translation. The recombinant E. coli expressed a truncated phase tail shear protein as expected.
In other words, if the stop codon was introduced into the cyanophage genome, it will become a virus that cannot release or infect cyanobacteria, thus preventing the escape of recombinant phage.
Subsequently, the fluorescent protein will be employed as indicators to validate codon expansion technology, and the expression of related genes would be optimized.

Outlook

• Next, the fluorescent protein will be employed as indicators to validate the codon expansion technology. The application of recombination cyanophage to infect cyanobacteria will also be explored.
• The model parameters will be identified and optimized to guide the application of our project in the real world.
• These results would be probably presented in our next year iGEM project.

Achievements

• We expect to construct recombinant cyanophage carrying the microcystin degrading enzyme to address the concern of algal blooms.
• The MlrAs which could degrade microcystin-LR with favorable catalytic activity have been characterized. The truncated phage tail protein has been successfully expressed in E. coli.
• The codon expansion technology would be applied to ensure the biosafety of recombinant cyanophage. Functional phage tail protein will only be expressed with the addition of unnatural amino acid Boc-lysine, which ensures that the reproduction of recombinant cyanophage is tightly controlled.

Integrated HP

In integrated HP, we interviewed researchers, experts, social organizations, and so on. By communicating with them, we learned about the severity of cyanobacteria problem and current solutions, which provided new ideas for our team’s projects.
We have fully analyzed the results of our HP. Through these, our project becomes responsible and good for the world.




In education & engagement, we carried out scientific publicity by offline lectures and live broadcasts.



In short, through education and engagement, we aim to let the public understand synthetic biology and the IGEM competition.

Acknowledgements and Sponsors

Acknowledgements
√ Finally we completed the train of thought design under the guidance of Professor Zhengjun Li and Professor Xinxiao Sun.
√ Professor Yigang Tong
√ Professor Xiaolin Shen
√ Mr. Cheng Li
√ Xiaoqi Niu
√ Mengru Wang
√ Huanhuan Fu
√ Funding and site support from Beijing University of Chemical Technology

Sponsors

References
[1] Bourne D G . Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR.[J]. Applied & Environmental Microbiology, 1996, 62.
[2] Ren YY. Synthesis, expression and purification of microcystin-degrading enzyme Mlr A gene optimized by Lactococcus lactis preference codon. [D]. Jilin University, 2015.
[3] Yane-Shih Wang, Xinqiang Fang, Ashley L. Wallace, et al. A Rationally Designed Pyrrolysyl-tRNA Synthetase Mutant with a Broad Substrate Spectrum[J]. Journal of the American Chemical Society, 2012.
[4] Daichi M , Shigeko K , Yoshihiko S , et al. Transcriptome Analysis of a Bloom-Forming Cyanobacterium Microcystis aeruginosa during Ma-LMM01 Phage Infection[J]. Frontiers in Microbiology, 2018, 9:2-.