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

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 , 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.

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:

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.

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.

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 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 ^[5] . 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 ^[6] .

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.

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:

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:

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.

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.