Team:Nanjing-China/Poster

Poster: Nanjing-China

About Us

Nanjing-China is an innovative and enterprising team with 18 members from School of Life Sciences and School of Chemistry and Chemical Engineering of Nanjing University. Our team originated from the scientific research and innovation training for undergraduates. We participate in the world's top synthetic biology competition, iGEM, to complete scientific research projects in fields of energy, environmental protection, medical biotechnology and other new applications, consequently improving the comprehensive practical ability of undergraduates and realizing the goal of personalized training.

PolyP Pro: Organic and inorganic biosynthesized hybrid materials

Presented by Team: Nanjing-China

Zhen Jiang¹, Yaxin Chen², Hanze Liu², Ziyang Xv², Mingchang Wang², Xiaohan Qian², Guangyu Hu², Shaoli Zhao², Yifei Bian², Yixuan Zhang², Zhongqi Xv², Shijie Li², Zhengni Wang², Jiankun Ren², Jiaqi Hu², Chaorui Ma², Jingran Qie², Yuxuan Xia², Pengpeng Zhang², Yuanyuan Ding², Wei Wei³, Xin Wang⁴, Xinru Pan⁵, Longqian Zhu⁵.

¹Student Leader.
²Student Member.
³Primary PI, ⁴Secondary PI, ⁵Instructors.

Project Abstract

There are many problems with synthetic chemical materials, such as the contamination and safety risks in production process. By means of Synthetic Biology, we hope to biosynthesis the inorganic material polyphosphates, which is commonly used in chemistry, with biomolecular proteins in order to obtain a new environmental friendly material, which not only maintains the original function of the biomolecule but also optimize its chemical properties.

Inspiration&Introduction

What happened?

Before our project began, a true story happened to our team member Zhongqi Xv attracted our attention to fire accidents. During a whale watching tour, he witnessed a fire accident in which not only did fierce smoke and burning flames devour a ship but also ignited the tarpaulin of Xu's boat. Although there was no danger in the end, the boat fire was put out in time and no casualty were caused, this experience of escaping from fire left Xu a deep expression, and initiated our thinking about the fire.

Fire, brings human light and energy, along with plenty of disasters, smoke, flame, tears. Around the world, fire has become a crucial security issue. So we come up with an idea of developing a novel fireproof material.

What's new?

Polyphosphate(polyP), which consists of four to one thousand phosphate groups and is a biomacromolecules with highest negative charge density, possessing many stable physicochemical properties such as fireproof capacity, is expected to protect proteins, so that the complex will have the ability to perform protein's biological functions under harsh conditions.

Our project will expand the application field of polyP, showing its marvelous performance in the field of synthetic biology, promote the research process of organic-inorganic hybrid materials, and our new method of using synthetic biology to realize hybridization could provide new insights for researchers in related fields around the world.

Our Value: Responsible and good for the world and mankind
(Human Practices)

Our project is consistent with our value

Interview with Gesanghua Paint Company: Our team members conducted interviews on the topic of fireproof coatings with Mr. Da Jiang, the manager of Gesanghua Paint company.



The results of our work show that traditional fireproof materials cannot meet the needs of market. Neither the production of fireproof materials is environmentally friendly nor the materials themselves possess desirable properties. Therefore, it is urgent to develop novel fireproof materials.

Interview with Prof. Wei: Professor Wei Wei said that the hybridization of organic-inorganic materials is currently a frontier research field, and related research is still in the exploratory stage. He suggested we start from the hybrid of an easily-characterized protein, for example, green fluorescent protein(GFP), and polyP as early exploration.

Our Goals

Our previous Human Practices work proved that our project can achieve the following goals:

1. Develop novel fireproof material to protect our world from fires
2. Use technology of synthetic biology to achieve pollution-free production process
3. Explore the unknown and promote scientific development

Design&Model: +36GFP
(Improvement of an existing part)

Supercharged GFP(+36GFP)

To achieve a better electrostatic interaction between GFP and polyP, we designed a brand new supercharged GFP(+36GFP) plasmid which possesses great amount of positive charges(since polyP is a biomacromolecules with highest negative charge density).

Modeling

We obtained the tertiary structure of +36GFP.

We simulated the binding patterns between polyP and +36GFP, and proved that multiple proteins could bind to one single polyP at different sites. Positively charged amino acids contribute a lot to the interactions.

Summary

In all, we constructed the 3D structures of +36GFP, gave potential instructions to the mutation of other target proteins, and revealed the binding patterns between polyP and +36GFP.

Build&Test: Our idea works!

+36GFP is successfully eluted and purified!

Hybridization succeeded!

We determined the phase separation boundary of +36GFP and polyP binding, selected the corresponding concentration in the phase separation boundary, and performed the binding experiment smoothly.

PolyP can stabilize biomolecules!

+36GFP combined with polyP showed much greater brightness compared to the single +36GFP(Fig1) and the effect of prolonged fluorescence is better in higher polyP's concentration(Fig2).



As the binding concentration of polyP increased, the fluorescence intensity of +36GFP increased and the thermal stability was also improved(Fig3). The fluorescence intensity of +36GFP binding with polyP in denaturing solution was lower than the initial fluorescence intensity(Fig4).



The above experiments were also demonstrated in cells. +36GFP and polyP can be synthesized seperately and combined successfully in E.coli cells.

Next Stage: Hybrid polyP with spidroin
(Human Practices)

At the time when the combination of + 36GFP and polyP is about to succeed, we interviewed Leqi Textile to broaden the application field of our "organic-inorganic biosynthesis new materials".



Mr. Zhen, the general manager of Leqi, affirmed our idea and suggested that we could try to combine polyP with spidroin, which is a new textile material with high toughness and strength. He believes it could be a promising new material for organic-inorganic biosynthesis.

Leqi textile's proposal highly promoted the progress of our project, enabling us to apply "novel organic-inorganic biosynthesis materials" to a wider range of fields.

Design&Model: Modify spidroin and simulate hybridization

In order to hybrid polyP with spidroin(sp, negatively charged) through electrostatic force, we changed the negatively charged amino acids and obtained two new proteins——spidroin1(sp1) and spidroin2(sp2).

Modeling of sp and sp2

We predicted the tertiary structures of sp(Fig5)&sp2(Fig6).



Besides, as we expected, the binding patterns between spidroins and polyP are similar to that of +36GFP, a few extra interesting results emerged:

1. The His tag was intentionally inserted into the proteins to ease the purification process. Some of our docking results indicate that it is also a promising docking site. It is possible that under appropriate pH conditions, His tag can undertake the job of binding to polyP.
   
   
   
   
2. The binding energy predicted by Autodock4 shows that while the binding energy between sp and polyP remains high, sp2 and +36GFP show lower binding energy which indicate strong interactions.
   
   

Summary

In conclusion, while natural spidroin(sp) is not a good candidate for forming strong interactions with polyp. Sp2, however, can theoretically form strong electrostatic interactions with polyP, indicating that the experiment was a valuable mutation trial.

Build&Test: Failure

We conducted a series of experiments for sp, sp1 and sp2. According to the SDS-PAGE detection results, sp and sp1 can be expressed in vivo, but the expression level of them were so low that the purification failed.

Though the expression level of sp2 in E.coli was relatively high, it was mainly present in the inclusion body, which made further tests impossible.

Learn from Human Practices work

We interviewed Prof. Cao and Prof. Tian from College of Engineering and Applied Sciences. Professor Tian gave us suggestions on protein purification and stressed the importance of buffer for successful protein elution.



Besides, professor Cao pointed out that reconstructing spidroin may affect its folding. Therefore, he proposed us a new idea and inspired us to combine spidroin with +36GFP to form a new fusion protein, and then hybrid the fusion protein with polyP.

Engineering Success!

Inspired by interviews with professors, we tried to fuse +36GFP and spidroin(sp). We attached +36GFP-SP fusion protein gene sequence to PET28a vector and obtained the recombinant plasmid PET28a-+36GFP-SP. The results showed that the fusion protein was successfully expressed and mainly existed in supernatant. The purification was also successful.



Due to the epidemic, we haven't conducted a thorough experiment with this fusion protein. But more experiments are still continuing.

Proposed Implementation

Based on our comprehensive investigation and the results of experiments and modeling, it is imperative to develop organic-inorganic biosynthesized material, and our project is feasible and promising.



On one hand, the researchers can use our ideas to refine the chemical property of target proteins or adopt our modeling approach to get a better understanding of molecular docking. On the other hand, the manufacturers and medical workers can produce more awesome materials by hybriding polyP with other biomolecules.

Safety Issues

We attended a seminar held by Genscript and confirmed that our project complies with safety regulations and is beneficial to the whole world and mankind.

Education

Due to the epidemic situation, we conducted series of online activities to promote synthetic biology and popularize our project.

Leading the Destiny of Life

On Nanjing University undergraduate admissions official platform, we launched a Science communication activity called "Leading the Destiny of Life " and broadcast live on other public platforms, totally receiving thousands of clicks.

Voice of Life: interview with researchers
(Opening Weekend Festival Activity)

To promote synthetic biology better and popularize the ideas of iGEM, we got in touch with Fudan, NJU-China, ZJU-China and organized ‘Voice of Life: interview with researchers’ in the Opening Weekend Festival.

Achievements&Acknowledgements

In all, we meet all these standards of Bronze, Silver and Gold medal.



We would like to express our sincere gratitude to all our team members for their hard-working and persistence under such a special epidemic condition. We are also grateful to iGEM Foundation for giving us this precious opportunity to know about synthetic biology and conduct this explorative project. Besides, we are fortunate to establish a close partnership with NAU-China, where we frequently exchange our ideas and mutually help each other. We are also thankful for all professors, interviewees, who helped us during our whole project.