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

This summer, CHINA_CD_UESTC team worked very hard in order to make a high-efficiency enzymatic biofuel cell (EBFC) by enriching the Laccase on the cathode electrode. We transferred four operons-- mamAB , mamGFDC , mamXY and mms6 , which were related to magnetosome formation into E.coli. the modified E.coli can produce Laccase-carried magnetosome. Therefore, we could immobilize and enrich Laccase on the cathode electrode by magnet. In our project, we improved previous laccase part ( BBa_K863005 ) and made it visible.

The EBFC schematic diagram as following is the final prototype of our project(Figure 1):

Figure 1 . Schematic diagram of our EBFC. On the anode, glucose is oxidized to gluconolactone, where the electrons are transferred from the GOX to CNT. On the cathode, Laccase is immobilized and enriched on the electrode by magnetosome. Electrons are transferred from CNT to Laccase where dioxygen is reduced to water.

After a review of the relevant literature ^[1] , we learned that the Laccase has advantages over other oxidases. Thereby, we chose the Laccase as the enzyme for cathode. In order to make Laccase visible, we designed a recombinant vector to fuse RFP with the laccase. And laccase gene was obtained from BBa_K863005 on the 2015 Kit Plate2. While the RFP gene was taken from BBa_E1010 on the 2015 Kit Plate3. We designed the vector piGEM-RL as below(Figure 2).After constructing the vectors completely, we detected whether it work or not by the method of ABTS ^[3] :

Figure 2. Schematic of piGEM-RL construction. Laccase: efficient oxidase, catalyzes the substrate to produce electrons and environmentally friendly. RFP: the reporter protein.

The Laccase can be used to construct a common EBFC as biological cathode. we conceived a prototype( Figure 3). In the EBFC, we used carbon paper which was full of carbon microfibers as electrode because it has good conductivity and large surface area. Glucose oxidase and RFP+Laccase were chosed to catalyze reaction for producing electricity!

Figure 3. Schematic diagram of our EBFC. On the anode, glucose is oxidized to gluconolactone, where the electrons are transferred from the GOX to CNT. On the cathode, electrons are transferred from CNT to Laccase where dioxygen is reduced to water.

The components of the EBFC are listed in the table 1.

The main materials of our EBFC.

The main materials of our EBFC(Figure 4). Carbon paper consists of carbon microfibers manufactured into flat sheets. It is used to help facilitates the reaction. Glucose oxidase oxidizes glucose into glucolactone. Electrons are released in the reaction.The fusion protein(RFP＋Laccase) transfers electrons to the terminal electron acceptor oxygen.

Figure 4. (A) Carbon papers as the electrode. (B) Glucose oxidase on the anode. (C) RFP＋Laccase on the cathode.

In order to immobilize and enrich Laccase on the cathode pole,we want to connect Laccase with magnetosome. Then the magnetosome carrying Laccase can be attracted by magnet. First, we need to obtain lots of magnetosomes.In the magnetotactic bacteria, there are four steps to generate magnetosome ^[5] :1-invagination, 2-protein localization, 3-initiation of crystal mineralization, 4-crystal maturation.There exist four operons-- mamAB , mamGFDC , mamXY and mms6 , which are related to magnetosome formation.

1. mamAB

This section describes the function of the vector piGEM-mamAB. It carries mamAB operon whose length is up to 17kb. Previous studies have shown that mamAB operon is one of the four core operons related to magnetosomes formation ^[9] . For consideration of the whole operon length (up to 17kb), compatibility and vector carrying capacity, we finally chose the vector pET-28a ^[6] as backbone. Accordingly, we put the mamAB operon into E.coli by the vector designed as followed:

Figure 5. Schematic of piGEM-AB construction.

Since the length of mamAB operon is up to 17kb, it is difficult to directly get its complete gene fragment. After studying the sequence, we divided mamAB operon into three parts which amplified from Magnetospirillum gryphiswaldense MSR-1 , and connected together by the following steps:

Figure 6. Schematic of the subclone method.

2. mamGFDC + mamXY + mms6

Previous study have shown that although the exact mechanism is not completely understood, these three operons are indispensable in modifying the formation of the magnetosome. Therefore, we built them on one vector to explore its practical effect modification ^[7] . Currently already known as following:

We need co-transfer the three vectors into E. coli , so the vector we chose be able to co-transformation with the vector pET-28a and pACYCDuet-1 is pCDFDuet-1. The final design of vector is shown in the following figure:

Figure 7. Schematic of piGEM-G6X construction.

We decided to get two gene fragments mamXY and mamGFDC + mms6 from Magnetospirillum gryphiswaldense MSR-1 genome. We respectively designed the method of gene obtain shown in the following figure:

Figure 8 . Schematic of the piGEM-G6X construction method.

3. Connection magnetosome with Laccase

Meanwhile, in order to make Laccase enriched, we designed a recombinant vector to fuse express mamW and RFP with the laccase. The protein MamW, a magnetosome transmembrane protein, can connect Laccase and magnetosome. The RFP reporter can make the novel structure visible. So we designed the vector piGEM-WRL. As the vector will be co-transferred with another two vectors, we chose the pACYCDuet-1 as the backbone.

Figure 9. Schematic of piGEM-WRL construction. The protein MamW is a magnetosome transmembrane protein ^[2], mamW gene was amplified from the Magnetospirillum gryphiswaldense MSR-1

In a word, we wanted to fix laccase on magnetosome membrane, and utilize the magnetotaxis of magnetosome to enrich laccase on the cathode.