Team:BNU-China/Improve
Improvement
Team UCLA in 2015 iGEM used honeybee silk protein 3 (part number: BBa_K1763000) in their project, which can serve the purpose of being the substitution of spider thread and natural silk. Although it is not as strong as other kinds of silk proteins, the honeybee silk protein has some advantages compared to spider thread and natural silk: the gene of honeybee silk protein is smaller, with no duplicated sequences, and easier to be synthesized and modified by molecular biology technology.
However, the silk protein produced from UCLA has no colors, thus with less aesthetic characteristic and uneasy to be observed directly. In order to observe the produced honeybee silk protein more directly, we add a blue chromoprotein, amilCP (part number: BBa_K592009) , to the end of the silk protein. amilCP is a blue chromoprotein comes from coral, which has a strong blue after expressing. The color has the highest absorption peak at around 588nm, which can be observed by naked eyes directly without any equipment.
We designed 2 pairs of primers for the genes of honeybee silk protein and blue chromoprotein by molecular biology technology. (There is a GGGGS linker in the middle of two fragments. ) Then we use infusion technology to insert two fragments after T7 promoter of pET21a(+) vector. The verification by PCR is showed in Figure 1. After then we introduced the recombinant plasmid into E.coli BL21(DE3). In 48 hours, there is blue chromoproteins expressed in bacterium solution, which can be observed by naked eyes (Figure 2). Therefore, the improvement of honeybee silk protein is practical and valid.
(Figure 1 verification of recombinant plasmid by PCR)
(Figure 2 the bacterium solution of expression group and control group)
The result of the experiment is showed in the following figures. Figure 3 showed the different colors of E.coli DL21 with different expression levels after centrifugation. There are largely expression group, slightly expression group and control group from left to right. The samples are investigated by SDS-PAGE after crushing the bacteria of three groups by high temperature. As it is shown in Figure 4 , after dyed by coomassie brilliant blue, there is a large number of proteins around expected 56-57kD from largely expression group, a small number of proteins from slightly expression group, and almost no significant expression from control group. The result of SDS-PAGE shows that the improved honeybee silk protein can be observed by naked eyes, and the different color shades of blue can reflect the expression level of the protein.
(Figure 3 the bacteria of different groups after centrifugation)
(Figure 4 the result of SDS-PAGE)
By infusing chromoprotein with different colors after the honeybee silk protein, we can produce silk protein with different colors, which will allow us to obtain the expression time, expression level and expression efficiency of the silk protein, therefore control the production of the honeybee silk protein efficiently. Not only can we make the silk protein more aesthetic, but also we can apply the silk protein into more areas widely, such as health and medicine, textile industry and biomaterial.
In improvement module, we provide the part named honeybee-blue protein (part number: BBa_K2220028).
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