Difference between revisions of "Part:BBa K4614002:Experience"
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| − | We constructed an expression vector of R5 and its surface display carrier protein IPN fusion protein, using T7 promoter as the promoter, and induced expression, and after reviewing the literature, we selected to induce 3 h at 37 °C at a final concentration of 1.0 ug/mL in the logarithmic phase, disrupted the bacteria, and performed Western blotting experiments on the supernatant and precipitation of the cell disruption solution to verify the expression of the protein of interest. | + | We constructed an expression vector of R5 and its surface display carrier protein IPN (encoded by Lnak) fusion protein, using T7 promoter as the promoter, and induced expression, and after reviewing the literature, we selected to induce 3 h at 37 °C at a final concentration of 1.0 ug/mL in the logarithmic phase<sup>[1]</sup>, disrupted the bacteria, and performed Western blotting experiments on the supernatant and precipitation of the cell disruption solution to verify the expression of the protein of interest. |
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| − | <img src="https://static.igem.wiki/teams/4614/wiki/si-parts/ | + | <img src="https://static.igem.wiki/teams/4614/wiki/si-parts/wb.png" width="500" height="auto" class="centered-image"> |
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<p class="figurelegend">Fig1.Bacterial holoprotein Western blotting development result</p> | <p class="figurelegend">Fig1.Bacterial holoprotein Western blotting development result</p> | ||
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We silicified the mutant strains using the method of silicification of bacteria obtained from the literature, and the control group did the same, we collected the silicified bacteria and observed the bacteria using transmission electron microscopy to obtain the silicification effect of R5 under the silicification conditions we used. | We silicified the mutant strains using the method of silicification of bacteria obtained from the literature, and the control group did the same, we collected the silicified bacteria and observed the bacteria using transmission electron microscopy to obtain the silicification effect of R5 under the silicification conditions we used. | ||
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| + | <img src="https://static.igem.wiki/teams/4614/wiki/si-parts/dianjing.png" width="600" height="auto" class="centered-image"> | ||
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| + | <p class="figurelegend">Fig2.Transmission electron microscope image | ||
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| + | According to transmission electron microscopy observations, we found that R5 anchored on the surface of E. coli, catalyzed the hydrolysis of TEOS into silica in the TEOS silicification system, and deposited on the bacterial surface, forming a silica shell on the R5-E. coli surface, while changing the permeability of the cell membrane, allowing TEOS to enter the cell and generate silicon-filled cells. The experimental group bacteria cells had normal morphology, were filled with silicon, and had intact and smooth cell walls. The control group bacteria cells had less intracellular material, incomplete cell walls, and varying degrees of deformation. To some extent, this indicates that bacterial silicification can maintain the cell shape and provide a certain rigidity. | ||
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| + | References of CAU_China | ||
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| + | [1]薛双红. 基于细菌表面展示技术的功能性无机材料合成研究[D].武汉理工大学,2019. | ||
===User Reviews=== | ===User Reviews=== | ||
Latest revision as of 13:19, 12 October 2023
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Applications of BBa_K4614002
We constructed an expression vector of R5 and its surface display carrier protein IPN (encoded by Lnak) fusion protein, using T7 promoter as the promoter, and induced expression, and after reviewing the literature, we selected to induce 3 h at 37 °C at a final concentration of 1.0 ug/mL in the logarithmic phase[1], disrupted the bacteria, and performed Western blotting experiments on the supernatant and precipitation of the cell disruption solution to verify the expression of the protein of interest.
Fig1.Bacterial holoprotein Western blotting development result
We silicified the mutant strains using the method of silicification of bacteria obtained from the literature, and the control group did the same, we collected the silicified bacteria and observed the bacteria using transmission electron microscopy to obtain the silicification effect of R5 under the silicification conditions we used.
Fig2.Transmission electron microscope image
According to transmission electron microscopy observations, we found that R5 anchored on the surface of E. coli, catalyzed the hydrolysis of TEOS into silica in the TEOS silicification system, and deposited on the bacterial surface, forming a silica shell on the R5-E. coli surface, while changing the permeability of the cell membrane, allowing TEOS to enter the cell and generate silicon-filled cells. The experimental group bacteria cells had normal morphology, were filled with silicon, and had intact and smooth cell walls. The control group bacteria cells had less intracellular material, incomplete cell walls, and varying degrees of deformation. To some extent, this indicates that bacterial silicification can maintain the cell shape and provide a certain rigidity.
References of CAU_China[1]薛双红. 基于细菌表面展示技术的功能性无机材料合成研究[D].武汉理工大学,2019.
User Reviews
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