Adhesion

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New parts involved:

• [1] pET-J23119 INP-silicatein + INP-YFP-csgA
  Fusion of Ice Nucleation Protein (INP), Silicatein, adhesion protein csgA and Yellow Fluorescent Protein (YFP) This 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.
  
  
• [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.
  
  

Other parts involved:

• [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).

Flushing Test

By integrating the ice nucleation protein (INP) gene with the CsgA gene, the bacteria can effectively attach to buildings and form biofilms capable of neutralizing the harmful effects of acid rain. Additional genetic modifications enable these microorganisms to produce enzymes that neutralize sulfuric acid and other corrosive agents, thereby maintaining the integrity of structural materials. To verify that E. coli can successfully adhere to glass slides despite buffer washing, indicating the production of adhesion proteins, we designed an adhesion test to measure the effects of washing at different pH solutions.

As depicted in Figure A1, 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, at pH 3 (Figure A1.1), both [1] and [3] show significantly high adhesion: [1] has higher CFU than BL21-pET11a (*** p < 0.001 ) and [7] (&&&& p < 0.0001 ), while [3] is significantly higher than BL21-pET11a (#### p < 0.0001 ) and [7] (!!!! p < 0.0001 ). At pH 5 (Figure A1.2), the adhesion of [1] and [3] is higher than [7] (&, ! p < 0.05 ) but lower than BL21-pET11a (**, # p < 0.01, p < 0.05 ). At pH 7 (Figure A1.3), [1] and [3] maintain superior adhesion compared to the control groups, with [1] significantly higher than BL21-pET11a (**** p < 0.0001 ) and [7] (&&&& p < 0.0001 ), and [3] significantly higher than BL21-pET11a (#### p < 0.0001 ) and [7] (!!!! p < 0.0001 ). Moreover, at pH 9 (Figure A1.4), [1] remains highly adhesive compared to BL21-pET11a (* p < 0.05 ) and [7] (&&&& p < 0.0001 ), while [3] is significantly higher than [7] (! p < 0.05 ) and shows no significant difference (ns) from BL21-pET11a.

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).

As shown in Figure A2, the flushing test results for strains pET11a, [1] pET-J23119 INP-silicatein + INP-YFP-csgA, [3] pET-J23119-INP-YFP-csgA, and [7] pET-T7-INP-YFP demonstrate that our engineered bacteria adhere most efficiently in a strong acidic environment pH 3. This is consistent with our hypothesis, as the pH of acid rain (~pH 4) falls within this acidic range.

Quantitatively, the adhesion efficiency of the CsgA-expressing strains [1] and [3] was significantly higher than that of the pET11a and [7] control strains across all tested pH levels. A sharp decline in adhesion was observed from pH 3 to pH 5 across all groups, suggesting that a pH below 5.0 strongly enhances CsgA-mediated adhesion. A one-way ANOVA confirmed that the differences in adhesion between the different plasmids were statistically significant (p < 0.0001), validating the critical role of CsgA in acid-resistant binding.

The data indicate that the integration of the ice nucleation protein (INP) and CsgA genes confers strong adhesive properties on the engineered bacteria in acidic conditions. This successful genetic strategy allows for their effective deployment on traditional building materials.

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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.

Prevention

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New parts involved:

• [5]pET-YhaM
  In order to convert cysteine, we use YhaM which plays a role in L-cysteine detoxification. By creating cysteine desulfhydrase, it converts cysteine into pyrovate, hydrogen sulfide and ammonium ions.
  
  
• [6]pET-T7-RBS-NSP4-sulfite reductase alpha-sulfite reductase beta
  We combined sulfite reductase alpha with sulfite reductase beta, which is capable of reducing sulfite efficiently to hydrogen sulfide and also reducing sulfate since it is overexpressed. By inserting the two genes into the MCS blocks (with a T7 promoter), the engineered bacteria is able to produce two types of enzyme in huge amounts simultaneously, providing multiple catalytic domains and enhancing efficiency.
  
  

Other parts involved:

• [4]pET-YdeD(BBa_K4171005)
  To eliminate sulfate and sulfite amounts on the building surface, we aim to remove and degrade the final product L-cysteine amounts to speed up the sulfate reduction pathway. We use YdeD (BBa_K4171005),which is a transmembrane protein that pumps L-cysteine (Cys) out of the cells.
  
  

Cysteine destruction test & Sulfate Reduction Test

Sulfate reduction test and cysteine ​​destruction tests were both designed to remove cysteine ​​and improve sulfate reduction efficiency. YhaM can degrade cysteine ​​content and convert it into H2S, pyruvate, and ammonium, accelerating the sulfate reduction process. The combination of sulfite reductase α and sulfite reductase β can also effectively reduce sulfite to hydrogen sulfide and, due to overexpression, can also reduce sulfate.

The ability of YdeD (BBa_K4171005) and YhaM to degrade cysteine was confirmed by comparing intracellular levels with and without a 2mM cysteine supplement. While all strains showed comparable baseline cysteine levels without supplementation, the control (BL21-pET11a) exhibited a significantly higher final cysteine concentration than the YdeD and YhaM strains after supplementation, demonstrating effective cysteine consumption by the engineered strains.

The sulfate and sulfite reduction tests were successful, with all engineered strains showing significant reductions compared to the control. Strain [6], featuring an extracellular sulfite reductase, demonstrated the highest reduction for both ions. While all engineered strains effectively reduced both sulfate and sulfite, their performance was notably more efficient with sulfite. This is likely because sulfite reduction is a more direct step in the cysteine biosynthesis pathway. These results confirm the ability of our engineered bacteria to reduce sulfate and sulfite under acidic conditions.

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1. Kopriva, S. and Koprivova, A. (2004) 'Plant adenosine 5’-phosphosulphate reductase: the past, the present, and the future,' Journal of Experimental Botany, 55(404), pp. 1775–1783. https://doi.org/10.1093/jxb/erh185.
2. 3. NEOGEN Corporation (2021) TOTAL SULFITE (Enzymatic) ASSAY PROTOCOL. report. https://prod-docs.megazyme.com/documents/Assay_Protocol/K-ETSULPH_DATA.pdf.

Repairing

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New parts involved:

• [1] pET-J23119 INP-silicatein + INP-YFP-csgA
  Fusion of Ice Nucleation Protein (INP), Silicatein, adhesion protein csgA and Yellow Fluorescent Protein (YFP) This 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.
  
  

Other parts involved:

• [2] pET-J23119-INP-silicatein
  By inserting the INP gene into our sequence, silicatein can be anchored on the cell surface to interact with extracellular precursors.

• [8] pET-T7-RBS-INP-silicatein-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.
  

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

Silica Formation Test

To identify and quantify the precipitates on the cell surface, a colorimetric assay was employed. The precipitates were treated with HCl and NaOH, after which any soluble silica was reacted with ammonium molybdate and ascorbic acid to form a blue complex, confirming its identity. The mass of silica was determined by measuring the optical density at 825 nm and referencing a standard calibration curve prepared with known silicon concentrations.

Successful silica catalysis was demonstrated by the experimental groups, which produced significantly higher biosilica levels than the controls (p < 0.01, ANOVA). All plasmids maintained stable production. Although strain [2] (pET-J23119-INP-silicatein) showed a slight performance advantage, the similar yields between strain [1] (which includes INP anchoring) and strain [8] (without specialized anchoring) indicate that extracellular localization of silicatein via INP does not increase silica formation. The lack of significant difference among the three experimental groups confirms that each system is capable of efficient and consistent silica production.

Silica Formation Test on Limestone

After confirming biosilica formation in culture, we assessed the practical application of our engineered *E. coli* on cracked cement. This experiment aimed to quantify the advantage of CsgA-mediated adhesion by comparing the silica formation on cement by strains expressing the CsgA protein against those that do not.

As shown in Fig. N8, the crack width was visibly reduced only after treatment with the [1] pET-J23119 INP-silicatein + INP-YFP-csgA strain in the presence of 4mM tetraethyl orthosilicate for 96 hours. In contrast, no noticeable change in crack size was observed for the other strains—[2], [8], and the control (BL21-pET11a).

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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