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

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 piGEM-pACYCDuet-1-laccase-mamW-RFP, 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 ^[4], 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.

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-pET28a-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:

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

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.

In order to further study the formation mechanism of the magnetosome’s shape and size, and control them, we constructed the vector piGEM-pCDFDuet-1-mamGFDC-mms6-mamXY 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:

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:

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.