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The previous studies showed that the enzymatic biofuel cell (EBFC) has more advantages on operation and function over the ordinary biofuel cell (BFC). EBFC has three main advantages: 1) High efficiency of energy conversion; 2) Green alternative energy; 3) Characteristics fitted the biosensor. We found two high-efficiency oxidases, glucose oxidase and laccase, which can be used in anode region and cathode region of EBFC respectively. At the same time, we learned that laccase have some other advantages like ^[3] : 1) It comes from natural bacteria or plants, so it’s friendly to environment; 2) It has high-efficiency oxidability; 3) Laccase can oxidize a broad range of substrates so that it can be used in sewage disposal. It is definitely the most suitable choice to put laccase on the cathode.

Therefore, we set out to transform the cathode with laccase. For the purpose of visualizing the location and concentration of laccase, we combined RFP with laccase. After that, we designed a way of enriching laccase on the cathode--using magnetosomes！

Along with the development of times and population growth, energy consumption is increasing rapidly. Up to now, the thermal power is the main source. According to BP Energy Outlook 2035 , we can find that the world's fossil fuel reserves are declining ^[4] , and the largest shift of shares give us an insight into the most likely shape of the future energy landscape! (Figure 1)

Figure 1. There have been some rapid shifts in fuel shares in power generation in the past: oil gaining in the 1960s and losing in the 1970s; nuclear picking up in the 1970s/80s and falling in the 2000s; gas rising through the 1990s and 2000s. In the Outlook, the largest shifts are the increase in the renewables share and the decline in the coal share ^[4] .

Among numerous renewable energy, bioenergy is a kind of clean renewable energy and a potential excellent substitute for fossil fuel. With the advance of biotechnology biofuel cell (BFC) which can convert the chemical energy of fuel into electric energy with enzyme or microbial tissue as a catalyst, has been researched widely.

Previous iGEM teams had done some studies about microbial fuel cell (MFC). iGEM13_Bielefeld-Germany made an Escherichia coli Fuel Cell platform to provide an efficient electron transfer from the bacteria to the electrode. iGEM14_LZU-China cloned a NO3-sensor sequence and riboflavin producing genes into Escherichia coli for anode and a gene coding chromate (VI) reductase Yief was cloned into E.coli for cathode. iGEM14_SCAU-China boosted up the level of intracellular NAD+ for higher electron transfer rate.

Biofuel cell is divided into microbial fuel cell and enzymatic biofuel cell (EBFC). EBFC is a special kind of fuel cell which uses organics as fuels and enzymes as catalysts. EBFC is generally separated into anode region and cathode region by proton exchange membrane. Fuels are oxidized under the action of enzyme in the anode region. Oxygen is reduced in the cathode region.

EBFC have broad application prospect, so we want to create a new type of device to develop the bioenergy.

The main configurations of enzymatic fuel cells involve bioanodes based on glucose oxidase, glucose dehydrogenase or lactate oxidase and biocathodes based on copper oxidases such as laccase, tyrosinase or bilirubin oxidase. This concept was initiated by Mano et al. who implanted microbioelectrodes based on osmium redox hydrogels, in a grape obtaining thus 2.4mW at 0.54v ^[5] .

Laccases is a kind of copper-containing oxidoreductase. In the reduction reaction, the electron from the oxidation is transferred to the other three copper ions. These ions form a trinuclearic cluster, which transfers electrons to the terminal electron acceptor oxygen ^[6] .

Meanwhile, laccase has the property of oxidizing a wide range of substrates e.g., phenolic compounds, so it can be used in sewage disposal. Our project used these two enzymes and transformed the cathode. We constructed the expression vector of RFP + laccase and transformed it into E. Coli. The red fluorescence produced by RFP can be used as an indication of laccase’s concentration and activity. According to the method of electron transfer, EBFC can be divided into electronic media electrodes and direct electrochemical electrodes. Considered that the latter has high catalytic efficiency and small restriction by environment, we tried to enrich the laccase on the cathode to enhance the redox potential of our EFBC.

Figure 2. An electrochemical phenol biosensor based on the immobilization of laccase (Lac) on the surface of copper capped magnetic core–shell (Fe3O4–SiO2) nanoparticles (MNPs) ^[7] .

We obtained the laccase from BBa_K863005 . Traditional chemical approaches ^[7] of fixing laccase may affect the activity of laccase and are toxicological. So we hoped to find a better method!

Of course using synthetic biological methods is a great idea to achieve our goal. Magnetotactic bacteria (MTB), a kind of bacteria that can be attracted by magnet, are a superexcellent choice for us. We noticed that MTB contains a fantastic structure-- magnteosome. It is a magnetic nano materials covered by biofilm. And the magnetosome is essential to magnetotaxis.After our investigation, we decided to connect laccase to the magnetosomes’ membrane to enrich them on the cathode surface.

Figure 3. Transmission electron microscopy images of several different MTB showing their distinctive cell and magnetosome crystal compositions and morphologies. Scale bars = 500 nm in bacterial images and 100 nm in magnetosomes images ^[8] .

After our investigation, we decided to connect laccases to the magnetosome's membrane to gather them on the cathode surface.

But there are two problems for us to solve. On one hand, MTB are anaerobic, it means they are hard to be cultured. On the other hand, it is difficult to modify them. So, we were aiming to construct a magnetosome expression system in E.coli to solve those problems. According to a paper in Nature nanotechonlogy , we confirmed that transferring four related operons can make other bacteria magnetotactic ^[9] .

Finally, we co-transferred all the vectors we constructed to make E.coli produce magnetosomes carrying laccase. The special magnetosomes would be used into our EBFC to improve the electron transfer efficiency!

Purpose:

One of the novelties of our human practices this year is the contact between enterprises and us. This is the first time for us that we try to have positive action to communicate with social enterprises, raising awareness in the society about iGEM and synthetic biology. We approached two biotechnology companies in order to achieve the following points:

1: Communicate with different kinds of social people and get some advice from them.
2: Spread iGEM to more individuals and groups who want to have a good understanding of synthetic biology.
3: As we want to apply our Magnetotatic E.coli into the biofuel cell, we would like to learn more about the market situation.
4: Initial contact to the Chengdu Biology Maker.

1.Anno Medical Devices Co.Ltd

Aug 12th 2015, Our team leaved for office of Anno Medical Devices Co.Ltd in Longhu times street, Pi County, in order to converse with Mr.Xu，Professor Lei and Engineer Xiang of the Chengdu Biology Maker.

From the biological perspectives, “If what you want to get is the production of Magnetotatic E.coli, magnetic nanoparticle, the key point is about controllability and verifiability of experimental results. If you expect potential products，raw materials and intermediate operation of each step are best to comply with biosafety. In terms of the competition, there might be short in technical conditions, however it’s a good way to think more about ideas,” Professor Lei put forward some personal opinions in regard to our project.

“I do look forward to seeing your Magnetotatic E.coli are able to apply to the disease detection and treatment. As the same time, I am willing to offer support on instruments and reagents, and help us to connect with other related enterprises for more communication about our project and iGEM,” manager of Anno Medical Devices Co.Ltd, Mr.Xu said.

Engineer Xiang hope strengthen the communication and cooperation between the college students and the Chengdu Biology Maker,” Not only the guidance of Project design but also experimental operation, our open source laboratory can satisfy the requirement. It is a good way to develop extensive cooperation by attending iGEM competition as Biological enthusiasts.

2.Jin Douyun Space Company

Aug 13th 2015， we arrived at the Jin Douyun Space Company with Manager Zhiyong Xu, and communicated with Manager Jianjun Deng and Sichuan Strategic Alliance of Medical Devices Innovation director Dan Li.

After Kaiyue Zhang briefly introduced iGEM competition and our project, Manger Deng indicated that the prospect of iGEM as top competition of Synthetic Biology would be inestimable, and strongly supported college students to attend this competition to make a little contribution to the national biology. He expected the results of our project and encouraged us to promote the project’s development in the level of product to dig more application value about ''Magnetotactic Escherichia coli''.

At the end of the communication, Director Li showed that he would bring the propaganda of iGEM to the Sichuan Strategic Alliance of Medical Devices Innovation and supported the development of Synthetic Biology in China.

Through the communications with biotechnology companies, we had completed the goal that to promote iGEM out of campus. But we will never stop and make people in mounting numbers to realize and recognize synthetic biology, then we can use this to improve our life.