Difference between revisions of "Team:CHINA CD UESTC/Description"

This year, we were planning to make a high efficiency enzymatic biofuel cell (EBFC). As we studied the present situation of EBFC ^[1,2] , which have the advantages of operation and function compared with ordinary chemical batteries. EBFC have four main merits: 1) High energy conversion efficiency; 2) green alternative energy; 3) good biocompatibility; 4) meet the energy needs of biosensor. And we found two main efficiency oxidases in electrode rooms of EBFC which are Glucose and Laccase. At the same time, we learned that Laccase have some other advantages like ^[3] : from natural bacteria or plants which friendly to environment, efficient oxidase, make use of the sewage to produce electricity which protect environment. 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 visualized the location and content of Laccase, we connected RFP with Laccase, which also reflected the influence of environmental conditions to Laccase. After that, we designed a way of enriched Laccase on the cathode which used magnetosome. Because of its limited presence in the organism and difficult to use, we put magnetosome into E.coli . And we transformed laccase into this E.coli and created the magnetotactic E.coli which can produce Laccase.

Along with the development of times and population growth, energy consumption is increasing rapidly, and up to now 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 shifts of share 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 of microbial fuel cell (MFC). iGEM13_Bielefeld-Germany made an Escherichia coli Fuel Cell platform to provide for an efficient electron transfer from the bacteria to the electrode. iGEM14_LZU-China cloned a NO3-sensor sequence and a riboflavin producing genes into Escherichia coli for anode and gene coding chromate (VI) reductase Yief was cloned into E.coli for cathode. iGEM14_SCAU-China boosted up the level of intracellular NAD+(H) for higher electron transferring rate.

Microbial fuel cell is divided into microbial fuel cell and enzymatic biofuel cell (EBFC). Enzymatic biofuel cells are special kind of fuel cells which use organics as fuels and enzymes as catalysts. EBFCs are generally separated into cathode and anode region by proton exchange membrane. Fuels, for example, glucose, is oxidized under the action of enzyme fuel in the anode region, electrons and protons reach the cathode through the external circuit and the proton exchange membrane respectively, and oxide is reduced in the cathode region.

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

The main configurations of enzymatic fuel cells involved 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 2.4 mW at 0.54 V ^[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 broad 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 to E. coli, the red fluorescence produced by RFP can be used as an indication of laccase’s concentration and activity. In general, according to the method of electrons transfer, EBFC can be divided into the use of electronic media electrodes and direct electrochemical electrodes. Given that the latter has high catalytic efficiency and small restriction by environment, we try to fix laccase on the cathode to improve 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!

Biological methods were the better choices to achieve our objective. Magnetotactic bacteria (MTB), a kind of bacteria that can be attracted by magnet, are a superexcellent choice for our EBFC. We noticed that MTB contains a fantastic structure, magnteosome, magnetic nano materials covered by biofilm. The magnetosome is essential to magnetotaxis.

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 t the magnetosome’s membrane to gather them on the cathode surface.

But there are two problems for us to solve. On the 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 electron transfer efficiency!