Team:JNFLS/Contribution
For the contribution part, we have two sections in the follows: 3D printed hardware and creating some new parts.
I. We made a 3D printed mask model for our project.
1.Softwares
3D mask design needs 3Dmax, Photoshop, ZBrush, keyshot and setuna softwares.
2.Design procedures
2.1 choose the headform to design the size and shape of the mask to help us shape better. Import a normal scale headform into 3Dmax software.
2.2 Based on the headform, fix points on the head to determine the approximate position and pattern of the mask on the face. The shape of the mask is initially constructed in the form of points and planes in 3Dmax, and then the model is exported to Zbrush software.
2.3 Use ZBrush software to adjust the style of mask to make the model more precisely, since ZBrush is not affected by the number of planes. It is also convenient to view the final style of the mask.
2.4 After the mask style is determined, use setuna software to intercept the styles of front and back of the mask, to see whether the style and function meeting the final desired model. After repeated comparisons and revision, the final model was formed.
2.5 after the model is determined, use Zbrush software to reduce the planes (the model file will be very large). In the process of plane reduction, it is necessary to note that the plane reduction should be carried out under the condition of not destroying the approximate shape, and the plane reduction should be carried out under the standard operation (the plane reduction is easy to cause bugs). Then export the obj file to 3Dmax.
2.6 Use 3Dmax software to transform the model into several formats, including STL format (3D printing in the later stage), FBX format (convenient for rendering the model in the later stage) and obj format (secondary optimization of the model).
2.7 Import FBX format into keyshot software for rendering, and assign the desired material color to the model. After the rendering, mask model is finished.
3.3D mask model printing
This 3D printed mask will be used as a model to acquire the bacterial cellulose with fixed shape when we put it into the culture medium of the E. coli transferred with the genes for BC production.
II. We created some new parts.
In this virus epidemic season, we are lucky to have the opportunity to do some experiments in the laboratory. Although there is a limitation on the number of people in the lab for preventing infection of virus, we cloned some genes involved in the bacterial cellulose synthesis. For creating parts, they were cloned into the plasmid pSB1C3, hoping they are useful contribution for future iGEM.
1.BBa_K3605001
This is the first part we created. It contains glucose hexokinase (GHK) with 969bp, originated from Gluconacetobacter xylinus CGMCC 2955. Using glucose, this enzyme catalyzes the formation of glucose-6-phosphate, which is the glycolytic intermediate and the origin of substrate synthesis for bacterial cellulose production.
Glucose hexokinase (GHK) was amplified using PCR method, and inserted into the vector pSB1C3. The result was identified in figure 1.
Fig.1. The result of Glucose hexokinase (GHK) gene cloning.
M: Marker; 1: PCR result of GHK; 2: Digestion of pSB1C3 containing GHK.
The software SnapGene viewer was used to analyze the sequence of GHK, its feature is showed as follows:
2.BBa_K3605002
This part contains two enzymes (2277bp), which is a polycistron in original organism. One of them is phosphoglucomutase (PGM), it can isomerize the glucose-6-phosphate (G-6-P) to glucose-1-phosphate (G-1-P), which is the second reaction for bacterial cellulose production from glucose. Another enzyme is UDP-glucose pyrophosphorylase (UGPase), it can catalyze the glucose-1-phosphate (G-1-P) to react with UTP, forming uridine-5’-phosphate-α-D-glucose (UDPG), which is the third reaction for Bacterial cellulose production from glucose.
These two enzymes were also amplified using PCR method, and inserted into the vector pSB1C3. Figure 2 shows the identification result.
Fig.2. The result of PGM and UGPase gene cloning.
M: Marker; 1: PCR result of PGM and UGPase; 2: Digestion of pSB1C3 containing PGM and UGPase genes.
The software SnapGene viewer was used to analyze the sequence of this part, its feature is showed as follows:
3.BBa_K3605003
This part is bacterial cellulose synthase (Bcs) complex (9102bp) from Gluconacetobacter xylinus. It contains 4 subunits BcsA, BcsB, BcsC and BcsD, which catalyzes the bacterial cellulose synthesis, using uridine-5’-phosphate-α-D-glucose (UDPG). This is the last reaction for Bacterial cellulose production from glucose. The reaction catalyzed by this enzyme is as follows:
Due to the long sequence (9102bp), this enzyme complex was synthesized directly and connected into pUC57 vector firstly, then it was inserted into the plasmid pSB1C3. The identification result is showed in Figure 3.
Fig.3. The gene cloning result of Bcs complex.
M: Marker; 1: Digestion of pUC57 vector containing Bcs complex; 2: Digestion of pSB1C3 vector containing Bsc complex.
The software SnapGene viewer was used to analyze the sequence of Bcs complex, its feature is showed as follows:
