Part:BBa_K4614002
Lnak-R5
We used T7 promoter to induce Lnak and R5(part BBa_K461400) expression. IPN,a protein encoded by Lnak，a carrier protein from the genome of Pseudomonas syringae,was used to display R5 on the surface of bacteria.
In use, the modified E. coli can be cultured to the logarithmic stage, and IPTG with a final concentration of 1.0 ug/mL can be added to induce 3 h, and protein expression can be completed
We constructed an expression vector to express 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[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.
Fig7.Transmission electron microscope image
According to the transmission electron microscope observation, we found that R5 is anchored on the surface of E. coli, in the siliconization system of TEOS, catalytic TEOS hydrolysis to silica, and deposited on the surface of the bacteria, formed silicon shell on the surface of R5-E.coli . Observing the morphology of the bacteria, we found that the bacterial cells in the experimental group were normal in morphology, the cell wall was complete and smooth, while the control group had incomplete bacterial cell walls and different degrees of deformation. To a certain extent, it shows that the silicon shell formed on the surface of the bacteria has a certain protective effect on the bacteria and provides 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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 529
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 529
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 529
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 529
Illegal NgoMIV site found at 72
Illegal NgoMIV site found at 405
Illegal AgeI site found at 679 - 1000COMPATIBLE WITH RFC[1000]
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