Team:CHINA CD UESTC/Design

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DESIGN

  We mainly designed three vectors respectively carrying mamW + RFP + laccase , mamAB and mamGFDC + mms6 + mamXY . Our purpose is to accomplish our magnetotactic E.coli and fix the laccase on the magnetosome membrane. Finally we put the magnetosomes carrying laccases into our enzymatic bio-fuel cell (EBFC).

Overview

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.

Visible laccase in EBFC

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 [2] :

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.

Express magnetosome in E.coli for immobilization

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 [3] :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 [4] . For consideration of the whole operon length (up to 17kb), compatibility and vector carrying capacity, we finally chose the vector pET-28a [5] 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 [6] . Currently already known as following:

  • 1) mamGFDC ：

    Crystal size and shape are mainly regulated by proteins encoded in the mamGFDC operon (composed of the genes mamC, D, F, and G ) and its deletion also leads to a reduction of the size of the magnetite magnetosome crystals [6,7] .

  • 2) mamXY ：

    The mamXY operon encodes proteins related to the magnetosome membrane ( mamY, X, Z , and ftsZ -like genes) and its deletion causes cells of Magnetospirillum to produce smaller magnetite particles with superparamagnetic characteristics [8] .

  • 3) mms6 ：

    The mms6 operon contains five genes ( mms6, mmsF, mgr4070, mgr4071 , and mgr4074 ) [9] that also appear to be involved in magnetite crystal shape and size.

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[10], 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.

Reference

[1] Cosnier S, Holzinger M, Goff A L, et al. Recent advances in carbon nanotube-based enzymatic fuel cells.[J]. Frontiers in Bioengineering & Biotechnology, 2014, 2:45.

[2] Zhang P. Test method for the laccase activity with ABTS as the substrate[J]. Textile Auxiliaries, 2007.

[3] Lohsse A, Ullrich S, Katzmann E, et al. Functional Analysis of the Magnetosome Island in Magnetospirillum gryphiswaldense: The mamAB Operon Is Sufficient for Magnetite Biomineralization[J]. Plos One, 2011, 6(10):: e25561.

[4] Lohsse A, Ullrich S, Katzmann E, et al. Functional Analysis of the Magnetosome Island in Magnetospirillum gryphiswaldense: The mamAB Operon Is Sufficient for Magnetite Biomineralization[J]. Plos One, 2011, 6(10):: e25561.

[5] Lee H Y, Khosla C. Bioassay-Guided Evolution of Glycosylated Macrolide Antibiotics in Escherichia coli[J]. Plos Biology, 2007, 5(2):e45-e45.

[6] Araujo A C, Abreu F, Silva K T, et al. Magnetotactic Bacteria as Potential Sources of Bioproducts[J]. Marine Drugs, 2015, 13(1):389-430.

[7] Scheffel A, Gärdes A, Grünberg K, et al. The major magnetosome proteins MamGFDC are not essential for magnetite biomineralization in Magnetospirillum gryphiswaldense but regulate the size of magnetosome crystals[J]. Journal of bacteriology, 2008, 190(1): 377-386.

[8] Ding Y, Li J, Liu J, et al. Deletion of the ftsZ-like gene results in the production of superparamagnetic magnetite magnetosomes in Magnetospirillum gryphiswaldense.[J]. Journal of Bacteriology, 2010, 192(4):1097-1105.

[9] Dorothée M, Anna Q, Hojatollah V, et al. Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle[J]. Proceedings of the National Academy of Science, 2010, 107(12):5593-5598.

[10]Kolinko I, Lohße A, Borg S, et al. Biosynthesis of magnetic nanostructures in a foreign organism by transfer of bacterial magnetosome gene clusters[J]. Nature nanotechnology, 2014, 9(3): 193-197.