To contribute to the iGEM community and provide a firm foundation for future iGEM teams, we verified and improved two existing iGEM parts, T7-RBS-silicatein and T7-INP-YFP. Using pET11a, we introduced these parts into the E. coli BL21 strain. Our approach to heritage preservation involved a three-part solution: prevention, repairing, and adhesion.
Please click the buttons below to show detailed information of the 2 improved parts:
- [7] pET-T7-INP-YFP (K523013)-T7 tag
It is designed by the iGEM Edinburgh 2011 team and improved by the BJRS team in 2018 as BBa_K2833009. This construct encodes a fusion protein consisting of the Ice Nucleation Protein (INP) and Enhanced Yellow Fluorescent Protein (EYFP), under the control of a T7 Lac promoter. It serves as a control in adhesion experiments, containing INP-EYFP but lacking the functional csgA gene. It shows significantly weaker adhesion than csgA-containing constructs (plasmids [1] and [3]) but moderately better retention than the empty vector control (pET-11a vector). - [3] pET-T7-INP-YFP(K523013)-csgA
CsgA, known for forming amyloid nanowires in E. coli biofilms, especially at low pH, which reinforces adhesion under acid rain's influence, making the engineered bacteria effective for adhering to the surface. This plasmid combines the BioBrick (BBa_K523013) designed by Edinburgh 2011 with CsgA (BBa_K1583003), featuring Ice-Nucleation Protein (INP) and Yellow Fluorescent Protein (YFP) under the T7 promoter. INP directs CsgA to the cell surface, promoting adhesion between bacteria and surfaces, while YFP serves as fluorescent marker for us to locate the protein under microscope. - [1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA
Fusion part of [2] pET-J23119-INP-silicatein (K1890001) and [3] pET-T7-INP-YFP(K523013)-csgA. his composite part contains the silicatein gene (BBa_K1890001), the csgA gene (BBa_K1583000), Ice Nucleation Protein (INP) and the Yellow Fluorescent Protein (YFP) gene (BBa_K1352004). The INP gene, which is a transmembrane gene, should carry silicatein, csgA and the YFP gene to the outer membrane of E. Coli. Silicatein, originating from the demosponge Suberites domuncula, catalyzes the formation of polysilicate such as silica. The adhesion protein csgA is a protein monomer which aggregates to form Curli fibres in natural biofilms of E. Coli., which creates adhesion with the addition of the INP gene. The YFP gene should glow in yellow colour in the fluorescent microscope with the addition of the INP gene, which creates convenience while doing experiments.
Original part:
Improved parts:
These parts are involved in adhesion.
To investigate the ability of our bacteria to neutralize sulfuric acid and other corrosive agents and their ability to adhere, we conducted an adhesion test. We prepared three bacterial strains with pH values of 3, 5, 7, and 9 for testing in four buffer solutions. First, we incubated the three bacterial strains and BL21-pET11a overnight. For strains 3 and 7, we added IPTG to induce the incubation. Then, we added 100 μL of bacterial solution to a glass slide and incubated it in a 37°C incubator for 30 minutes. We then rinsed the slides with 1000 μL of H₂SO₄ (pH 3.5) and NaOH (pH 7.9), respectively, and observed them under a microscope. Results are shown below(Fig. C1 & C2)
As depicted in Figure C1, the flushing test results reveal that bacteria engineered with both CsgA and INP(BBa_K1890001) genes ([1], [3]) exhibit markedly superior adhesion to the surface compared to the control groups BL21-pET11a and [7]. Notably, in figure A1, at pH 3, both [1] and [3] have significant high adhesion ( p < 0.001 to p < 0.0001). In figure 1C, at pH 7, closer to neutral, superior adhesion of [1] and [3] compared to the control groups ( p < 0.0001). Moreover, in figure 1D, at pH 9, both [1], [3] remained high adhesion compared to [7] and BL21-pET11a, with the significant t-test result (p < 0.05 to p < 0.001). Nevertheless, at pH 5 (figure A. 1B), the adhesion of [1], [3] displayed higher CFU than [7] ( p < 0.05 ) but lower than BL21-pET11a ( p < 0.05 to p < 0.01 ).
At pH 3, the environment is more acidic, leading to protonation of the gatekeeper residues ( aspartic acids ) of csgA to reduce negative charges on the proteins, contributing to form effective fibril and higher adhesion. In contrast, as the pH increases to 5, the gatekeeper residues would shift more towards deprotonation, leading to higher electrostatic repulsion that slows fibril formation and weakens adhesion. Interestingly, at pH 7 and pH 9, buried residues shift towards deprotonation, while other residues likely remain protonated, maintaining stable fibril formation, higher adhesion than pH 5 and lower than pH 3. It is demonstrated that adhesion at pH 4 follows the same mechanism with pH 3, although adhesion at pH 4 is slightly lower than pH 3, it is significantly higher than at pH 5. (Bhoite et al., 2023).
Based on the data, our engineered bacteria—utilizing a combined solution of the ice nucleation protein (INP) and CsgA genes—demonstrated strong, effective adhesion in acidic conditions. This was quantitatively confirmed through adhesion assays (Fig. C1 & C2), where our engineered strains retained the highest number of colonies after rinsing with a strong acidic solution (pH 3 H₂SO₄). These results validate that our engineered bacteria can be successfully applied to the surfaces of traditional buildings for efficient adhesion.
1. Francis DM, Page R. Strategies to optimize protein expression in Escherichia coli. Curr Protoc Protein Sci. 2010 Aug;Chapter 5(1):5.24.1-5.24.29. doi: 10.1002/0471140864.ps0524s61. PMID: 20814932.
2. Bhoite SS, Kolli M, Chapman MR, Mukhopadhyay S. Electrostatic interactions mediate the nucleation and growth of a bacterial functional amyloid. Front Mol Biosci. 2023 Jan 11;10:1070521. doi: 10.3389/fmolb.2023.1070521. PMID: 36756360.
[8] pET-T7-RBS-silicatein (K1890001)-T7 tag
It is designed by iGEM TU Delft 2016 team and successfully expressed in E coli. by iGEM USAFA 2024 team. By inserting the two genes into the pET vector system, the engineered bacteria can catalyze silica from monomeric silicon compounds on their cell surface.[2] pET-J23119-INP-silicatein (K1890001)
By inserting the INP gene into our sequence, silicatein can be anchored on the cell surface to interact with extracellular precursors.[1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA
Joint part of [2] pET-J23119-INP-silicatein (K1890001) and [3] pET-T7-INP-YFP(K523013)-csgA
Original part:
Improved parts:
Recombinant protein production associated growth inhibition results mainly from transcription and not from translation[1]. Hence, when designing plasmid [2]INP-silicatein, we removed the strong RBS (BBa_B0034) for translation from BBa_K1890001 to test whether the translation of INP-silicatein functions as well as [8]RBS-INP-silicatein.4
These parts are involved in repairing.
Silica Formation Test
We designed and conducted a test whose aim is to determine whether the precipitates on the cell surface are silica. Precipitates can be seen under microscope but we need to testify its identity and quantify it. After being treated with HCl and NaOH, silica should react with ammonium molybdate and ascorbic acid to form a blue color. If so, the precipitate can be proved to be a silicon compound, thus proving it to be silica. We also plot a calibration curve between known silicon concentration and the OD value of 825nm. Samples are dissolved into solution and the OD value is tested to deduce the mass of silica catalysed.Results are shown in Fig. C1
Silica Formation Test on Limestone:
Following the successful demonstration of silica formation in bacterial cultures, we evaluated its application in a real-world context. Using cracked limestone obtained from a hydraulic machinery sample, we tested whether our engineered E. coli, enhanced with the CsgA adhesion protein, could form silica on its surface. The aim of this test was to determine if the CsgA protein leads to greater silica formation on limestone compared to strains without it.
Fig. C4 demonstrates that only the [1] pET-J23119 INP-silicatein + INP-YFP-csgA strain, when supplemented with 4mM tetraethyl orthosilicate, produced a noticeable narrowing of the cement crack after 96 hours. The other experimental groups ([2] and [8]) and the control group (BL21-pET11a) showed no measurable change in crack dimensions.
1. Okorie, Ngozi, et al. MOLYBDENUM BLUE METHOD DETERMINATION of SILICON in AMORPHOUS SILICA. 2015, www.semanticscholar.org/paper/MOLYBDENUM-BLUE-METHOD-DETERMINATION-OF-SILICON-IN-Okorie-Momoh/973b1f72180060d896ebbf900da4a5fe4c2555a7. Accessed 2 Oct. 2025.
2. Schröder, Heinz C, et al. “Acquisition of Structure-Guiding and Structure-Forming Properties during Maturation from the Pro-Silicatein to the Silicatein Form.” Journal of Biological Chemistry, vol. 287, no. 26, 1 June 2012, pp. 22196–22205, https://doi.org/10.1074/jbc.m112.351486. Accessed 14 Sept. 2023.
3. Vigil, Toriana N., et al. “Surface-Displayed Silicatein-α Enzyme in Bioengineered E. Coli Enables Biocementation and Silica Mineralization.” Frontiers in Systems Biology, vol. 4, 30 May 2024, https://doi.org/10.3389/fsysb.2024.1377188. Accessed 2 Oct. 2025.
4. Zhaopeng Li , Ursula Rinas: Recombinant protein production associated growth inhibition results mainly from transcription and not from translation