Difference between revisions of "Team:CHINA CD UESTC/Design"
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<img src="https://static.igem.org/mediawiki/2015/3/3b/CHINA_CD_UESTC-DesignPlus02.png" width="40%"> | <img src="https://static.igem.org/mediawiki/2015/3/3b/CHINA_CD_UESTC-DesignPlus02.png" width="40%"> | ||
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Figure 3. Glucose enriched on the anode. | Figure 3. Glucose enriched on the anode. | ||
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Figure 4. Laccase ＋ RFP enriched on the cathode. | Figure 4. Laccase ＋ RFP enriched on the cathode. | ||
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Revision as of 18:37, 13 September 2015
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DESIGN
We mainly designed three vectors respectively carrying laccase + mamW + RFP, mamAB and mamGFDC + mamXY + mms6. The purpose is to accomplish our magnetotactic E.coli with laccase and put them into our enzyme bio-fuel cell (EBFC). let's have a detailed view in the design process.
Overview
This summer, CHINA_CD_UESTC team made a high-efficiency enzymatic biofuel cell (EBFC) by constructing Magnetotactic E.coli which can produce Laccase. In order to achieve this goal, we co-transferred a gene coding the fusion protein which connected Laccase with MamW. The protein MamW which is a magnetosome transmembrane protein as a connection between magnetosome and Laccase. Therefore, we can immobilize Laccase on the magnetosome membrane (MM). And we also transferred four operons – mamAB , mamGFDC , mamXY , mms6 - which are related to magnetosome's formation into E.coli . Once we put the magnetotactic E.coli which produce Laccase onto the cathode, can we utilize the magnetotaxis to gather oxidases which will efficiently improve the efficiency of EBFC. By this reason, we designed vectors and experimental process as three parts: mamW + RFP + Laccase , mamAB and mamGFDC + mamXY + mms6 .
We designed an enzymatic biofuel cell (EBFC) schematic diagram as following which was 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 to the Laccase with oxidation, which can be put into the cell cathode. At the same time, we hope to use the reporter gene RFP to help locate and content the Laccase protein visualized. In addition, by changing the environment of Laccase, we can better understand its optimum environmental conditions used by the visualization of RFP . According to the principle of co-transformation, we designed the vector pACYCDuet-1, and we put together the genes of Laccase and RFP to the vector.
The main role of each gene as follows:
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(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.
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(2) RFP: The reporter gene, which protein 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 MSR-1 extracted genomic by PCR. 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.
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
Construction of enzymatic biofuel cell (EBFC)
As we conceived the prototype of EBFC and read the literature of constructing EBFC [1] , we prepared materials of components of our Laccase EBFC as following: (100ml)
Table 1. Components of the EBFC.
We put large and rough surface area carbon paper (Fig. 2) 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. 3), got the Laccase as described above and bought the carbon paper. As we prepared everything already, we successfully constructed a basic EBFC.
Figure 2. Carbon papers on both anode and cathode.
Figure 3. Glucose enriched on the anode.
Figure 4. Laccase ＋ RFP enriched on the cathode.
mamAB
In the magnetotactic bacteria, there are four steps to generate magnetosome [1] : 1-invagination, 2-protein localization, 3-initiation of crystal mineralization, 4-crystal maturation. Thus, in our project design, we constructed two vectors which responsible for implementing the further modification of the magnetosome formation.
This section will describe the function of the vector piGEM-mamAB. It carried mamAB operon which region up to 17kb located in MTB genome. Prior studies have shown that mamAB operon is not only one of the four core formation unit related to magnetosome, but also responsible for generating the basic structure of magnetic body [2] . Compared to those three operons which modified the formation of magnetosome, mamAB relatively independent to complete its work that produced a fairly complete magnetosome. Accordingly, we put this fatal functional unit mamAB into E.coli by the vector designed as following:
For consideration of the gene cluster size (17kb), compatibility and vector carrying capacity, we finally chose the backbone vector pET28a [3] .
Since mamAB operon lengthen out to 17kb, it is difficult to directly get its complete gene fragment for us. After studying their sequence, we divided mamAB operon into three parts which amplified by PCR from the genome of magnetotactic bacteria MSR-1 , and connected together through 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.
Reference
[1] 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
[2] 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
[3] 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
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 [1] . Currently already known as following:
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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 [2]
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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 [3,4] .
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3.mms6：
The mms6 operon contains five genes (mms6, mmsF, mgr4070, mgr4071, and mgr4074) [5] that also appear to be involved in magnetite crystal shape and size.
In brief, magnetosomes produced by magnetotactic bacteria 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:
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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.
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2.Origin
We select the CDF ori as vector’s replication origin.
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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.
Reference
[1] 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
[2] 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
[3] 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
[4] 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
[5] 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
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.
