Part:BBa_M36515
Silaffin 1 Peptide Encoding Gene
This gene codes for a peptide called silaffin 1. It is composed of silaffin 1A1, silaffin 1A2, and silaffin 1B peptides. This gene is naturally found in marine diatoms, which use silaffin in order to incorporate silica into various cellular structures. The silaffin 1 peptide precipitates silica when isolated from an organism and reacted with silicic acid.
Contribution From CAU_China 2023
Group: CAU_China, 2023 https://2023.igem.wiki/cau-china/
Author: Fen miaodi Zhu wanying Xu linjie
Summary:We did sequence optimization of this protein, and 57bp was selected from 795 bp and codon optimization was carried out, which achieved similar function to the original，and this sequence is called R5.
Improvement
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, 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[1], and after reviewing the literature, we selected to induce 3 h at 37°C at a final IPTG 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[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 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.
[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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 360
Illegal SapI.rc site found at 426
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