Part:BBa_K4614000

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

In this part, the principle of R5 as TEOS hydrolysis and silica aggregation lies in the positive electricity of its Lys catalytic group^[2], in order to verify the rationality of this principle, we try to use acid instead of R5 to provide positive electric groups, and use different concentrations of hydrochloric acid or 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 form silica gel, and TEOS of 2 and 3 was catalyzed to generate silica gel after 84 hours.

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

Next, we constructed an expression vector to express R5 and its surface display carrier protein IPN fusion protein, and induced expression, and after reviewing the literature, we selected to induce 3 h at 37°C IPTG at a final concentration of 1.0 ug/mL in the logarithmic phase^[1], disrupted the bacteria, and the supernatant and precipitation of the cell disruption solution were used to progress 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^[3]. 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.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.

[2]Ping H , Poudel L , Xie H , Fang W , Zou Z , Zhai P , Wagermaier W , Fratzl P , Wang W , Wang H , O'Reilly P , Ching WY , Fu Z . Synthesis of monodisperse rod-shaped silica particles through biotemplating of surface-functionalized bacteria. Nanoscale. 2020 Apr 30;12(16):8732-8741. doi: 10.1039/d0nr00669f. PMID: 32307501.

[3] Lee, L. J. (1989). Modeling and computer simulation of reactive processing. Comprehensive Polymer Science and Supplements, 20, 575-617. https://doi.org/10.1016/B978-0-08-096701-1.00238-X