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

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<P>同时，为了解决基因太大无法直接获取的问题，我们决定从趋磁细菌MSR-1的基因组中分别获取mamXY 和  GFDC+mms6 两个基因片段，设计了如下图所示的方法，最后连接在一个载体pCDFDuet-1上。</P>
 
<P>同时，为了解决基因太大无法直接获取的问题，我们决定从趋磁细菌MSR-1的基因组中分别获取mamXY 和  GFDC+mms6 两个基因片段，设计了如下图所示的方法，最后连接在一个载体pCDFDuet-1上。</P>
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<P>通过初步的检测手段，包括酶切验证和测序，结果均显示，我们成功的连接了这两个基因片段，并成功构建了该载体。</P>
 
<P>通过初步的检测手段，包括酶切验证和测序，结果均显示，我们成功的连接了这两个基因片段，并成功构建了该载体。</P>

Revision as of 18:16, 7 September 2015

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DESIGN

  We are a skillful and persistent group of nine Finns. We started as a group of students who didn't really know each other, assuming that we were going to spend our summer studying synthetic biology with strange colleagues. In the end we got a bunch of new friends and (in addition to studying synthetic biology) we just might have spent one of the best summers of our lives.

Laccase + mamW + RFP

After a review of the relevant literature, 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 [1] . Thereby, we came up to the laccase with oxidation, which can be put into the cell cathode and better immobilized by magnetosome. At the same time, we hope to use the reporter gene RFP to help locate and content the mamW protein visualized. According to the principle of co-transformation [2] , we designed the vector pACYCDuet-1, and we put together the genes of laccase + mamW + RFP to the vector.

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) mamW: The main function is membrane localization, found in magnetic bodies outside the membrane vesicles, which can help laccase immobilization. And mamW is related to the formation of magnetosome.

(3) RFP: The reporter gene, which protein 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.

After constructing this vector completely, we encountered some troubles. Detecting by ABTS method [3] , the three genes laccase + mamW + RFP did not work as we expected.Thus, we had designed three other vectors in order to validate our initial vision contain the following genes:

(1) laccase + RFP: Visualizing the expression situation and contents of laccase by RFP.

(2) mamW + RFP: Visualizing the contents and specific location out of the vesicle membranes of mamW protein by RFP, and verified whether mamW protein play a major role in the formation of magnetosome or not.

(3) 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.

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

mamGFDC+mamXY+mms6

为了能够进一步对磁小体的形状、大小进行控制，以及深入研究其形成机理。我们构建了载体iGEM-pCDFDuet-1-mamGFDC-mms6-mamXY，它包含了磁小体的三个重要操纵子，mamGFDC,mamXY,mms6。



以前的研究表明，尽管具体机制尚不完全清楚，但这三个操纵子对磁小体的形成有不可或缺的修饰作用[1]所以我们将其构建在一个载体上，去探究其修饰作用的实际效果。目前已知

  • 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]

  • 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] .

  • 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.

简言之，这三个操纵子的缺失将会致使趋磁细菌产生的磁小体无法成链，这将极大影响其趋磁性。因此，我们决定组件化这部分基因。并最终提交了两个相关基因的parts。分别是BBa_K1779100，BBa_K1779101。

我们选择了pCDFDuet-1 作为其载体，主要是出于以下三点考虑：

  • 1.兼容性。

    我们共有3个载体需要转入大肠杆菌，所以选的载体要能够与载体pET28a和pACYCDuet-1共转化

  • 2.起始位点。

    CDF ori

  • 3.承载力。

    操纵子大小为10.4kb，质粒必须能够承载这种尺寸的基因。

最终的载体设计如图所示：

(1) laccase + RFP: Visualizing the expression situation and contents of laccase by RFP.

同时，为了解决基因太大无法直接获取的问题，我们决定从趋磁细菌MSR-1的基因组中分别获取mamXY 和 GFDC+mms6 两个基因片段，设计了如下图所示的方法，最后连接在一个载体pCDFDuet-1上。

通过初步的检测手段，包括酶切验证和测序，结果均显示，我们成功的连接了这两个基因片段，并成功构建了该载体。

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