Team:CHINA CD UESTC/Design

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DESIGN

  We mainly designed three vectors respectively carrying mamW+RFP+laccase , mamAB and mamGFDC + mamXY+ mms6. 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 made a high-efficiency enzymatic biofuel cell (EBFC) by constructing novel magnetotactic E.coli which can produce laccase. In our EBFC, laccase is necessary for electrons transfer, we improved previous laccase part (BBa_K863005) to make it visible . On the one hand, we transferred four operons-- mamAB , mamGFDC, mamXY and mms6, which are related to magnetosomes' formation to make E.coli magnetotactic. On the other hand, we constructed a vector which can express a fusion protein (mamW+RFP+laccase). The protein MamW, a magnetosome transmembrane protein, is a connection between magnetosome and laccase. Therefore, we can immobilize laccase on the magnetosome membrane (MM).

The EBFC schematic diagram as following is our prototype of the project:

Figure 1. Schematic diagram of EBFC. At the anode, glucose is oxidized to gluconolactone, where the electrons are transferred from the GOX to CNT. Catalase decomposes hydrogen peroxide into oxygen and water. At the cathode, electrons are transferred from CNT to laccase where dioxygen is reduced to water.

RFP + Laccase

After a review of the relevant literature [1] , we learned that in the previous bio-fuel cells, the applications of enzyme fuel cell is very wide, and magnetotactic bacteria can generate the magnetosome attracted by magnet. Thereby, we came up with the method that put the laccase into the cell cathode. At the same time, in order to make laccase protein visible we hope to use the reporter gene RFP to locate and mark it. In addition, by changing the environment of laccase, we can find out its optimum environmental conditions using the visualization of RFP. So we designed the vector piGEM-R-Lac.

The main role of each gene as follows:

  • (1) Laccase: Efficient oxidase, catalyzes the substrate to produce electrons, which can be used as a biological cathode in enzyme fuel cell and applied in batteries.

  • (2) RFP: The reporter protein which can locate and content the protein MamW visualized out of the vesicle membrane.

After constructing this vector completely, we detected whether it work or not by the method of ABTS [2] and got the positive result. Furthermore, we learned from literature [3] that mamW gene which located in magnetosome genome had the function of membrane localization. mamW was found in magnetic bodies outside the membrane vesicles, which can help Laccase immobilization. And mamW is related to the formation of magnetosome. Therefore, we would like to connect mamW to the working vector. However, the fusion expression of these three proteins may had a little difficult, which no one had studied and done before, so we designed the following two vectors:

(1) mamW + laccase : fixed the expressional Laccase in the cell cathode and verified whether MamW protein play a major role in the formation of magnetosome or not.

(2) mamW + RFP + laccase : Based on the above vector, RFP protein also can locate and content the MamW protein visualized out of the vesicle membrane, while the contents and expression of Laccase.

Wherein, mamW gene was amplified from the Ms.gryphiswaldense MSR-1 . 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.

Construction of enzymatic biofuel cell (EBFC)

As we conceived the prototype of EBFC and read the literature of constructing EBFC [4] , we prepared materials of components of our Laccase EBFC as following: (100ml)

We put large and rough surface area carbon paper (Fig. 2A) on both anode and cathode in order to facilitate the attachment of Laccase. The implementation of functional prototype was not just the materials mentioned above, we also bought the Glucose (Fig. 2B,2C), got the Laccase as described above and bought the carbon paper. As we prepared everything already, we successfully constructed a basic EBFC.

Figure 2. (A)Carbon papers on both anode and cathode.(B)Glucose enriched on the anode.(C)Laccase＋RFP enriched on the cathode.

mamAB

In the magnetotactic bacteria, there are four steps to generate magnetosome [4] :1-invagination, 2-protein localization, 3-initiation of crystal mineralization, 4-crystal maturation. Thus, in our project design, we constructed piGEM-AB which is responsible for the formation of magnetosomes.


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[5]. Compared to the other three operons which modify the formation of magnetosomes, mamAB can complete its work--producing a basic magnetosome independently. Accordingly, we put the fatal functional operon mamAB into E.coli by the vector designed as followed:

For consideration of the operon length (up to 17kb), compatibility and vector carrying capacity, we finally chose the backbone vector pET-28a [6].

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 MSR-1, and connected together by the following steps:

As we preliminary verified our vector by means of enzyme digestion and sequencing, the results have shown that we have successfully connected the three gene fragments, and the vector was successfully constructed.

mamGFDC + mamXY + mms6

In order to further study the formation mechanism of the magnetosome’s shape and size, and control them, we constructed the vector piGEM-G6X which including three important operons of magnetosome: mamGFDC , mms6 and mamXY .


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:

  • 1. mamGFDC ：

    Crystal size and shape are mainly regulated by proteins encoded in the mamCD 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 [8]

  • 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 [9,10] .

  • 3. mms6 ：

    The mms6 operon contains five genes ( mms6 , mmsF , mgr4070 , mgr4071 , and mgr4074 ) [11] that also appear to be involved in magnetite crystal shape and size.

In brief, magnetosomes produced by MTB cannot form a chain without those three operons, which will extremely affect its magnetotaxis. So we decided to componentize the genes of this part. And finally we submitted two parts of related genes which are BBa_K1779100 and BBa_K1779101 .

We chose pCDFDuet-1 as our vector, mainly for the following three considerations:

  • 1. Compatibility

    We need a total of three vectors into E. coli , so the vector we chose be able to co-transform with the vector pET28a and pACYCDuet-1.

  • 2. Origin

    We select the CDF ori as vector's replication origin.

  • 3. Carrying Capacity

    Due to the large size of the operon which is 10.4kb, the plasmid must capable to carry this size of gene.

The final design of vector is shown in the following figure:

Meanwhile, in order to solve the problem that gene is too large to be directly obtained, we decided to get two gene fragments mamXY and GFDC + mms6 from MSR-1 genome. We respectively designed the method of gene obtain shown in the following figure. The last one, mamW was connected on the vector pCDFDuet-1.

As we preliminary verified our vector by means of enzyme digestion and sequencing, the results have shown that we have successfully connected the two gene fragments, and the vector was successfully constructed.

The promoter verification

In order to find the reason why the magnetosome was not formed in the E.coli , we constructed several vectors to investigate the operons’ promoters. We chose pSB1C3 as backbone, and replaced the PlacI of the part BBa_J04450 or replaced RFP which was the first genes of every operons.

Reference

[1] Serge Cosnier, Michael Holzinger, Alan Le Goff (2014). “Recent advances in carbon nanotube-based enzymatic fuel cells.” Bioengineering and Biotechnology 2:45, doi: 10.3389/fbioe.2014.00045

[2] Zhang Peng (2007). “Test method for the Laccase activity with ABTS as the substrate.” China Academic Journal Electronic Publishing House 24:1

[3] Isabel Kolinko, Anna Lohße, Sarah Borg, et al. (2014). “Biosynthesis of magnetic nanostructures in a foreign organism by transfer of bacterial magnetosome gene clusters.” Nature Nanotechnology 9: 193-197, doi:10.1038/nnano.2014.13

[4] Ana Carolina V. Araujo 1, Fernanda Abreu 1, Karen Tavares Silva 1,2, Dennis A. Bazylinski 3 and Ulysses Lins 1,* Magnetotactic Bacteria as Potential Sources of Bioproducts. Mar. Drugs 2015, 13, 389-430; doi:10.3390/md13010389

[5] Anna Lohße1, Susanne Ullrich1, Emanuel Katzmann1, Sarah Borg1, Gerd Wanner1, Michael Richter2,Birgit Voigt3, Thomas Schweder4, Dirk Schu¨ ler1*.Functional Analysis of the Magnetosome Island in Magnetospirillum gryphiswaldense: The mamAB Operon Is Sufficient for Magnetite Biomineralization

[6] Citation: Lee HY, Khosla C (2007) Bioassay-guided evolution of glycosylated macrolide antibiotics in Escherichia coli. PLoS Biol 5(2): e45. doi:10.1371/journal.pbio.0050045

[7] Ana Carolina V. Araujo; Fernanda Abreu; Karen Tavares Silva; Dennis A. Bazylinski; Ulysses Lins. Magnetotactic Bacteria as Potential Sources of Bioproducts.Mar. Drugs 2015,13,389-430

[8] Scheffel, A.; Gärdes, A.; Grünberg, K.; Wanner, G.; Schüler, D. The major magnetosome proteins MamGFDC are not essential for magnetite biomineralization in Magnetospirillum gryphiswaldense but regulate the size of magnetosome crystals. J. Bacteriol. 2008, 190, 377–386

[9] Lohße, A.; Ullrich, S.; Katzmann, E.; Borg, S.; Wanner, G.; Richter, M.; Voigt, B.; Schweder, T.; Schüler, D. Functional analysis of the magnetosome island in Magnetospirillum gryphiswaldense: The mamAB operon is sufficient for magnetite biomineralization. PLoS One 2011, 6, doi:10.1371/journal.pone.0025561

[10] Ding, Y.; Li, J.; Liu, J.; Yang, J.; Jiang, W.; Tian, J.; Li, Y.; Pan, Y.; Li, J. Deletion of the ftsZ-like gene results in the production of superparamagnetic magnetite magnetosomes in Magnetospirillum gryphiswaldense. J. Bacteriol. 2010, 192, 1097–1105

[11] Murat, D.; Quinlan, A.; Vali, H.; Komeili, A. Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. Proc. Natl. Acad. Sci. USA 2010, 107, 5593–5598