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Applications of BBa_K4614000

In this part, the principle of R5 as TEOS hydrolysis and silica aggregation lies in the positive electricity of its Lys catalytic group, in order to verify the rationality of this principle, we try to use acid instead of R5 to provide positive electric groups, we use different concentrations of hydrochloric acid and oxalic acid, add different proportions of TEOS, observe and record experimental phenomena.

1）hydrochloride

Fig1.Silicification system of hydrochloride

Fig2.Silicification results of hydrochloride

In the siliconization experiment of hydrochloric acid, after mixing according to the above ratio for 16 hours, TEOS is hydrolyzed to form silica gel.

2）oxalic acid

Fig3.Silicification system of oxalic acid

Fig4.Silicification results of oxalic acid（after 72h）

Fig5.Silicification results of oxalic acid(after 84h)

In the oxalic acid-catalyzed siliconization experiment, after 72 h of mixing the system, TEOS in group 1 was catalyzed to generate silica gel, and TEOS of 2 and 3 was catalyzed to generate silica gel after 84 hours.

Based on the above results, we justify the principle of R5 silicification to a certain extent.

Next, we constructed an expression vector of R5 and its surface display carrier protein IPN fusion protein, and induced expression, and after reviewing the literature, we selected to induce 37 h at IPTG 37°C at a final concentration of 1.0 ug/mL in the logarithmic phase, disrupted the bacteria, and the supernatant and precipitation of the cell disruption solution were Western blotting, which verified the expression of the target protein.

Fig6.Western blotting development results of bacterial whole protein extraction

After that, we build a model to guide the siliconization time to a certain extent according to the silica concentration on the surface of the bacteria. According to the results, it can be seen that within 48 iterations, the silicon concentration diffuses rapidly, and with only 30% of the catalytic center, the silicon concentration in the reaction plane can basically reach saturation. In the experiment, we chose the catalytic time of 48 hours, and the actual silicon concentration iteration will definitely be shorter than one hour. This model can explain to a certain extent that when the silicification time is reached, silica has been relatively fully precipitated on the surface of E. coli.

Finally, we silicified the mutant strains using the method of silicification of bacteria obtained from the literature, and the control group also performed the same treatment, we collected the bacteria after siliconization, observed the bacteria using transmission electron microscopy, and obtained the silicification effect of R5 under the silicification conditions we used.

Fig7.Result of Western Blotting

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.