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Validation
The validation part is to verify our plasmids' nucleic acid quantification and confirm presence of protein.
All 8 plasmids are tested:
- [1]pET-J23119 INP-silicatein + INP-YFP-csgA
- [2]pET-J23119-INP-silicatein
- [3]pET-T7-INP-YFP-csgA
- [4]pET-J23119-ydeD(K4171005)
- [5]pET-J23119-yhaM
- [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta
- [7]pET-T7-INP-YFP (K523013)-T7 tag
- [8]pET-T7-RBS-INP-silicatein (K1890001)-T7 tag
qPCR
Aim:
To measure gene expression levels and assay performance.
Protocol:
In order to quantify gene expression, we used TRIzol to isolate RNA from bacterial broth. Then, reverse transcription is conducted to obtain cDNA from RNA. Lastly, qPCR is conducted.
Result:
All the 8 plasmids show greater expression of the target RNA expression than that of the control.
Conclusion:
This test indicates a higher target gene expression level of all 8 plasmids than that of the control.
Western Blot
Aim:
To confirm the presence of proteins in our samples.
Protocol:
1. Gel Electrophoresis (PAGE): To separate proteins by size as they migrate through a porous gel using an electric current.
2. Protein Transfer (Blotting): The separated proteins are transferred from the gel to a solid membrane using an electric current.
3. Membrane Blocking: The membrane is incubated with a blocking solution to prevent antibodies from binding non-specifically to the membrane.
4. Primary Antibody Incubation: The blocked membrane is incubated with a primary antibody that is specific to the target protein.
5. Secondary Antibody Incubation: A secondary antibody, which is linked to an enzyme or fluorescent tag, is added to bind to the primary antibody.
6. Detection and Visualisation.
Result:
The above data shows the successful Western Blot result of [1]pET-J23119 INP-silicatein(50.2kDa)+INP-YFP-csgA, [2]pET-J23119-INP-silicatein (50.2kDa), [3]pET-T7-INP-YFP-csgA (75.2kDa) and [5]pET-J23119-yhaM (45.3kDa).
Conclusion:
This test indicates the successful protein expressions of our plasmids.
Adhesion
The adhesion part is testing for the following 3 plasmids:
- [1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA
Silicatein (BBa_K1890050) , originating from the demosponge Suberites domuncula, catalyzes the formation of polysilicate. By adding the INP gene(BBa_K1890053) to silicatein, silicatein can be anchored on the cell surface to interact with extracellular precursors and catalyse the formation of polysilicate such as silica. Ice-nucleation protein (INP) allows the colonies to adhere firmly to the surface and not easily fall off, and it also helps anchor silicatein on the cell surface to facilitate extracellular silica formation. In addition, csgA can enhance the adhesion of the protein binding domain to surfaces at low pH, and the YFP design allows us to observe the colonies under a microscope. - [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. - [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
Aim:
To ensure the E.coli creates adhesion between the building surface, we utilized the csgA gene which produces fibre for the biofilm structure of bacteria, making it a good source for adhesion and it makes the adhesion even stronger in an acidic environment.
Data expected:
The flushing test confirmed that plasmids [1], [3], [7], and the control pET-11a vector successfully adhered to the slide surface.
As expected, the results indicate that the CsgA gene is expected to be crucial for strong adhesion. We expect bacteria carrying csgA gene (plasmids [1] and [3]) should adhere much more strongly than the controls(BL21-pET11a) and plasmid [7].A substantial number of cells remained attached even after rinsing with solutions of varying pH levels.
The data are also expected to suggest that acidic conditions (low pH) enhance the binding efficacy of the CsgA protein, as would be indicated by strong resistance to a pH 3.0 rinse.
In contrast, the control (pET-11a vector) and plasmid [7], which lack a functional csgA gene, are predicted to display only weak adhesion, with most cells being easily washed away.
Data results:
(*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns = not significant)
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).
According to Figure A2, the flushing test shows that the adhesion efficiency of [1] INP-silicatein-INP-YFP-csgA and [3] INP-YFP-csgA is generally higher than pET11a and [7] INP-YFP controls in pH3, pH5, pH7 and pH9. We also observed that the adhesion is highest at low pH(pH3) and drops by a lot from pH3 to pH5 across all groups. We suggest that pH below 5.0 strengthened csgA's adhesion activity. Additionally, our one-way ANOVA test indicates that the differences among plasmids were statistically significant (p < 0.0001).
BL21-pET11a control has higher CFU than others without flushing, which is possibly due to operational error. Nevertheless, the CFU of control has low colony count when flushed at different pH levels, which suggests that the control bacteria has weak adhesion ability.
Without flushing The number of colonies of this plasmid was lower than that of the control group (BL21-pET11a). After flushing when we rinsed the bacteria, the most colonies stayed when the pH was 3. This number was way higher than when the pH was 5, 7, or 9, showing it superior adhesion on surfaces, which was further demonstrated in the test of “Silica formation on limestones” in the Repairing results.
Similar to plasmid [1], the number of colonies of no flushing was also lower than the control group (BL21-pET11a). After rinsing, the number of retained colonies of plasmid [3] was the highest among all tested pH levels. This highest retention count was notably greater than the number of retained colonies observed when the pH was set to 5, pH 7, and pH 9. Overall, [1] and [3] both have great adhesion efficiency, while [3] has higher efficiency at pH 3 than [1] . Since the size of recombinant protein might negatively affect expression (Francis DM, Page R, 2010), it is reasonable that the plasmid with only INP-YFP-csgA is more effective at low pH.
The colony count of no flushing was lower than the control but higher than the plasmids [1][3]. After flushing, its colony count decreased more than the plasmids[1][3] but less than the control. For this plasmid, the colony numbers after flushing also showed some variation with pH: more colonies were present at pH 3 than at pH 5, and more at pH 9 than at pH 7. This control also exhibits low adhesion efficiency as the colony number after flushing is much less than ½ of [1] and [3] with csgA across pH levels.
The red circles in Figure A3, A4, A5 and A6 mark the images taken after flushing with pH 3 solution. The plasmids [1],[3],[7] and control BL21-pET11a retained the most colonies in a strong acidic environment after rinsing with H2SO4 at pH 3, demonstrating our high adhesion. This confirms that more colonies were found in more acidic environments, and so on, with the least colonies remaining in more alkaline environments.
Conclusion:
Based on the experimental data, our engineered bacteria exhibited strong, pH-dependent adhesion. The adhesion was most robust in highly acidic environments, peaking at around pH 3, while a noticeable decrease was observed at pH 5 due to higher electrostatic repulsion that slows fibril formation[2]. We demonstrated that the fusion of the ice nucleation protein (INP) gene with the CsgA gene created an effective system, enabling successful and controllable bacterial adhesion to traditional building surfaces under targeted acidic conditions.
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
The prevention part is testing for the following 3 plasmids:
- [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. - [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 [1] 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.
Mechanism:
In order to reduce sulfate and sulfite amounts on the building surface, we discovered the sulfate reduction pathway in bacteria. The final product of the pathway L-Cysteine is a useful amino acid in bacteria. Inspired by the equilibrium in chemistry, we brainstormed an idea of removing the final product of the pathway L-Cysteine to speed up the sulfate reduction pathway as the concentration of the product L-Cysteine decreases. Therefore, we use the YdeD gene to transport cysteine out of the bacteria and we use the YhaM gene to convert cysteine into hydrogen sulfide, pyruvate and ammonium ions.
To eliminate sulfate and sulfite amounts on the building surface, we aim to remove and convert 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. In order to convert cysteine, we use YhaM which plays a role in L-cysteine detoxification. By creating cysteine desulfhydrase, it converts cysteine into pyruvate, hydrogen sulfide and ammonium ions.
To test the mechanism of our engineered E. Coli., the functional tests are splitted into 4 parts:
- Cysteine desulfhydrase enzymes quantifying with qPCR
- Sulfate and sulfite reduction test
- L-Cysteine destruction test
- Hydrogen sulfide formation test
Cysteine desulfhydrase enzymes quantifying with qPCR
Aim:
A qPCR analysis of cysteine desulfhydrase enzymes was conducted to validate that their expressions increased after [4]pET-J23119-ydeD (K4171005) and [5]pET-J23119-yhaM lowered the cysteine concentration. The enzymes we tested included CysA, CysC, CysH, CysDN, and CysIJ.
Protocol:
We followed the qPCR protocol, using specific primers for each of the seven genes involved in sulfate reduction pathway, namely CysA, CysC, CysH, CysD, CysN, CysI and CysJ, respectively. [1]pET-J23119 INP-silicatein + INP-YFP-csgA is the control for the test.Result:
The results show that the expression levels of all cysteine desulfhydrase enzymes in the sulfate reduction pathway in [4]pET-J23119-ydeD (K4171005) were significantly higher than in both [1]pET-J23119 INP-silicatein + INP-YFP-csgA (control) and [5]pET-J23119-yhaM, while the expression levels of enzymes in [5]pET-J23119-yhaM are at similar levels to the control group. All of the p-values of T-Test between [1]pET-J23119 INP-silicatein + INP-YFP-csgA and [5]pET-J23119-yhaM were higher than 0.05, which means there were no significance.
The primary reason for the markedly higher expression of enzymes in [4]pET-J23119-ydeD (K4171005) is that [4]pET-J23119-ydeD (K4171005) is a transmembrane protein that directly pumps L-cysteine out of the cells. Since cysteine is transported outside the cell, there is little to no cysteine to be used for synthesizing other essential products in bacteria. Therefore, a compensatory sulfate response is triggered to produce cysteine for the bacteria, leading to a three- to six-fold increase of all the enzymes.
The cysteine desulfhydrase enzymes in [5]pET-J23119-yhaM have similar expression levels to the control because [5]pET-J23119-yhaM speeds up L-cysteine conversion to hydrogen sulfide, pyruvate and ammonium. This mechanism lowers the cysteine concentration inside the cell but increases the concentration of its degradation products. The need for a compensatory sulfate reduction to produce cysteine is less urgent than in [4]pET-J23119-ydeD (K4171005), so the expression of these genes remains at basal levels.
Pre-experiment: bacterial growth test
Aim:
Before the reduction tests, pre-experiments were performed to test whether 10μM, 100μM, and 1000μM sulfuric acid, sodium sulfate, sodium sulfite will affect BL21-pET11a growth. The results are shown below, indicating the concentration of sulfate does not have an effect on bacterial growth.Result:
Conclusion:
This pre-experiments test indicates the concentrations of sulfate and sulfite in the tested range do not have a significant effect on bacterial growthSulfate and sulfite reduction test
Aim:
To test whether our plasmids reduce sulfate and sulfite amount.Protocol:
In order to investigate the sulfate and sulfite reduction efficiency of our engineered bacteria, we used a Sulfate Assay Kit from Sigma-Aldrich and a Total Sulfite Assay Kit from Megazyme to conduct reduction tests. These kits allow us to measure the concentration of sulfate and sulfite accurately. We repeated each experiment three times.
After adding 1mM sulfuric acid solution and 1mM sodium sulfite to our engineered bacteria cultures respectively, we incubated them for 5 hours at 37°C. We then added IPTG in the sulfite reductase group at a 1:1000 dilution, and subsequently incubated the solution for 48 hours at 25°C. Sulfate and sulfite concentrations were then measured using the respective assay kits.
Sulfite group
For the sulfite group, ice-cold 1 M perchloric acid was first added to deproteinise the solution. The sample was then centrifuging at 12000rpm for 15 minutes, followed by adding 1 M NaOH to the supernatant to adjust the pH value to approximately 8 [4]. Lastly, we used a Total Sulfite Assay Kit(enzymatic) from Megazyme to determine the concentration of sulfite. Here is the mechanism of the total sulfite assay kit: Sulfite is oxidised to sulfate by the enzyme, sulfite oxidase, in the presence of oxygen (Shown in Figure P7.).
Hydrogen peroxide formed in this reaction is reduced in the presence of the enzyme, NADH peroxidase and NADH (Shown in Figure P8.).
The amount of NAD+ formed in the above reaction pathway is stoichiometric with the amount of sulfite. The consumption of NADH is measured by the decrease in absorbance at 340 nm. According to the kit protocol, we added and mixed the following reagents well in a cuvette: 250 µL of solution 1 (buffer), 100 µL of solution 2(NADH), 5 µL of sample solution, 125 µL of distilled water, 10µL solution. The absorbance at 340nm was measured using a spectrophotometer after an incubation of 4 minutes. Then, 10 µL suspension 4(sulfite oxidase) was added and mixed. The absorbance at 340nm of the solution was measured again after 30 minutes. The sulfite concentration of each sample was calculated based on the change in absorbance subsequently [4].
Sulfate group
For the sulfate group, TCA reagent was added to deproteinise the solution, followed by centrifuging at 14000rpm for 10 minutes. A Sigma-Aldrich Sulfate Assay Kit was subsequently used to determine the sulfate concentration of each sample. This method is based on the quantitative formation of insoluble barium sulfate in polyethylene glycol. The turbidity measured at 600 nm is proportional to sulfate level in the sample. We added 200 µL of supernatant into separate wells of a 96 well plate, and 100 µL of the master reaction mix was pipetted to each of the appropriate wells preferably using a pipette and mix well. The reaction was incubated at room temperature for 5 minutes and the optical density of the samples at 600 nm was measured [5].
Result:
Overall, the sulfate and sulfite reduction tests were successful. The concentrations of sulfate and sulfite in almost all of our engineered bacterial cultures showed significant decreases compared to the control (BL21-pET11a).
The sulfite concentration in cultures of [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM and [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta was significantly lower than in the control group (BL21-pET11a) after adding 1mM sodium sulfite into each culture for 48 hours. The sulfite concentration in [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta broth exhibited the most substantial decline. This is primarily attributed to the presence of an extracellularly exposed sulfite reductase, while the sulfite reduction performed by [4]pET-J23119-ydeD (K4171005) and [5]pET-J23119-yhaM occurs intracellarly. Both [4]pET-J23119-ydeD (K4171005) and [5]pET-J23119-yhaM demonstrated efficient sulfite reduction, with final concentrations below 0.4m.
With previously adding 1mM sulfate, the sulfate concentration in control(BL21-pET11a) culture was higher than that of [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM and [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta culture. Sulfate reduction in culture of [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta was also the most remarkable, which we attribute to the same extracellular mechanism. Furthermore, sulfate concentration in [5]pET-J23119-yhaM culture showed a noticeable drop, while that in [4]pET-J23119-ydeD (K4171005) broth had no significance.The concentration of sulfate in the BL21-pET11a broth remained unchanged. The sulfate reduction test proves the functionality of our plasmids in facilitating sulfate reduction.
The engineered bacteria performed notably more efficient sulfite reduction than sulfate reduction. We posit that it is mainly because the step of sulfite reduction is closer to cysteine in the metabolic pathway, making its reduction more direct and potent.
Comparing with the result of sulfate reduction pathway enzymes qPCR, the sulfate and sulfite reduction performed by [5]pET-J23119-yhaM was more significant than that of sulfate and sulfite. We believe it is primarily because mRNA expression often does not correlate to protein expression [6]. The qPCR test validates that [4]pET-J23119-ydeD (K4171005) removing cysteine does accelerate the expressions of those enzymes.
Conclusion:
The sulfate and sulfite reduction tests indicate that our engineered bacteria are able to reduce sulfate and sulfite under acidic conditions. Based on above data, [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta culture is capable to reduce sulfate and sulfite directly, whereas, [4]pET-J23119-ydeD (K4171005) and [5]pET-J23119-yhaM accelerate sulfate and sulfite reduction by lowering cysteine amount.Cysteine destruction test
Aim:
To test whether [4]pET-YdeD(BBa_K4171005) & [5]pET-YhaM reduce amount of cysteine.Protocol:
We used a Cysteine (Cys) Content Assay Kit, Colorimetric Method to test cysteine amount in [4]pET-YdeD(BBa_K4171005), [5]pET-YhaM and control. We measured the Cysteine level by doing pre-experiment tests with control(BL21-pET11a)for the optimal concentration of Cysteine to test for bacteria, ensuring both bacteria survival and appropriate amount of cysteine to be tested. The concentration of cysteine, optimal for experiments, is 2mM.Result:
Before adding extra cysteine, the cysteine amounts of the control group (BL21-pET11a), YdeD (BBa_K4171005) and YhaM are nearly the same. After additional 2mM cysteine, the cysteine level of control group (BL21-pET11a) is obviously greater than that of YdeD (BBa_K4171005) and YhaM.
Conclusion:
This test indicates the successful cysteine destruction of YdeD and YhaM.After testing the higher concentration of Cysteine desulfhydrase, we proved that YhaM does have the capability to produce Cysteine converting enzymes. However, to show the mechanism of how YhaM works, we must test the increased amount of hydrogen sulfide when sulfate and sulfite are added. Therefore, we did the following experiments for further proof of mechanism of the plasmids in the prevention part.
Hydrogen sulfide formation test
Aim:
To test whether [5]pET-YhaM reduces cysteine into hydrogen sulfide.Protocol:
We used a Hydrogen Sulfide Content Assay Kit to test amount of hydrogen sulfide in [5]pET-YhaM and control. We add Cyteine into LB broth and let it incubate with our bacteria overnight. Then we use the kit to test hydrogen sulfide amount. We measured the hydrogen sulfide level by doing pre-experiment tests with control(BL21-pET11a)for the optimal concentration of Cysteine to test for bacteria, ensuring both bacteria survival and appropriate amount of Cysteine to be tested. The concentration of Cysteine, optimal for experiments, is 8mM.Result:
(See Figure P13.) With previously added 8mM Cysteine, the hydrogen sulfide amount of YhaM is higher than that of the control group BL21-pET11a.
Conclusion:
This test indicates the successful hydrogen sulfide formation from Cysteine of YhaM.1. Wikipedia contributors. (2025, August 18). Sulfite reductase. Wikipedia. https://en.wikipedia.org/wiki/Sulfite_reductase
2. Kopriva, S., & Koprivova, A. (2004). Plant adenosine 5’-phosphosulphate reductase: the past, the present, and the future. Journal of Experimental Botany, 55(404), 1775–1783. https://doi.org/10.1093/jxb/erh185
3. Nakatani, T., Ohtsu, I., Nonaka, G., Wiriyathanawudhiwong, N., Morigasaki, S., & Takagi, H. (2012). Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli. Microbial Cell Factories, 11(1). https://doi.org/10.1186/1475-2859-11-62
4. NEOGEN Corporation (2021) TOTAL SULFITE (Enzymatic) ASSAY PROTOCOL. report. https://prod-docs.megazyme.com/documents/Assay_Protocol/K-ETSULPH_DATA.pdf.
5. Sulfate Assay Kit (MAK132) - Technical Bulletin. (n.d.). sigma-aldrich.com. Retrieved October 6, 2025, from https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/424/788/mak132bul.pdf?srsltid=AfmBOopdP1E2PepNHDzwP70_zKj9xnA-sJbLSo2BIEOW0A4p58EydTpT
6. Koussounadis, A., Langdon, S. P., Um, I. H., Harrison, D. J., & Smith, V. A. (2015). Relationship between differentially expressed mRNA and mRNA-protein correlations in a xenograft model system. Scientific Reports, 5(1). https://doi.org/10.1038/srep10775
Repairing
The repairing part is testing for the following 3 plasmids:
[1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA
Silicatein, originating from the demosponge Suberites domuncula, catalyzes the formation of polysilicate. This biobrick contains the short version of the silicatein gene. The gene is fused to the transmembrane domain of ice nucleation protein (INP) from Pseudomonas syringae. The coding sequence in this BioBrick is set downstream of the strong RBS BBa_B0034. Ice-nucleation protein (INP) allows the colonies to adhere firmly to the surface and not easily fall off, and it also helps anchor silicatein on the cell surface to facilitate extracellular silica formation. In addition, csgA can enhance the adhesion of the protein binding domain to surfaces at low pH, and the YFP design allows us to observe the colonies under a microscope.[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 (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.
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.
Silicon Molybdenum Blue Colorimetric Method
Aim:
To confirm the viability of our plasmids for expressing silicatein, we aim to determine whether biosilica can be successfully catalyzed by silicatein and to quantify its production. We employ a silicon molybdenum blue colorimetric method to examine silica formation and measure its mass, using tetraethyl orthosilicate (TEOS) as the precursor.
Mechanism:
TEOS is added to bacteria samples and they are incubated overnight. Silica samples produced by cultured bacteria are collected by centrifugation. The samples then undergo the first centrifugation to separate cell pellets from other components in the samples. The cell pellets are moved, and the samples (the supernatants) then undergo a second centrifugation to further precipitate silica, in which the supernatants are removed. Suspected silica should stick to the bottom, and the precipitate mass is the estimated value of the silica produced.
Silica samples are dissolved in NaOH. The resulting solutions are acidified to a pH of 1-2. They then react with ammonium molybdate, which provides molybdate ions (MoO₄²⁻). Under acidic conditions, the soluble silicon compounds react with the molybdate ions to form a yellow-colored acid complex known as silicomolybdic acid. To prevent interference from phosphates, oxalic acid is added before reduction. A reducing agent (ascorbic acid is used here) is then added, and it reacts with molybdenum(VI) (Mo⁶⁺) in silicomolybdic acid to form molybdenum(V) (Mo⁵⁺), which is blue in color.
The absorbance of the blue color can then be measured, confirming silica formation. To further quantify the amount of silica, a calibration curve is plotted between known silicon concentration and absorbance. By finding the corresponding concentration for the samples from their absorbance, the amount of silica can be calculated and the difference in production among different plasmid groups can be compared.
Data expected:
Control group (BL21-pET-11a) samples are expected not to show a denser blue color formation which would indicate larger silica formation than the experimental groups, since they do not contain silicatein and are thus unable to catalyze silica from the precursor. Moreover, [1]pET-J23119 INP-silicatein + INP-YFP-csgA and [2]pET-J23119-INP-silicatein are expected to show denser blue color formation than [8]pET-T7-RBS-INP-silicatein (K1890001)-T7 tag, as they express INP which anchors silicatein on the cell surface and increases the chance for silicatein to react with extracellular TEOS.
Results for plotting the calibration curve between silicon concentrations and absorbance:
Conclusion:
According to Figure R1, the data acquired from the test shows an expected linear pattern, verifying a concrete relation between the sample's silicon concentration and its absorbance, which is useful for analyzing unknown samples.
Results for silicon molybdenum blue colorimetric assay:
Based on the ANOVA and t-test analysis of the absorbance, this experiment conclusively confirms the successful enzymatic formation of biosilica from TEOS by silicatein constructs. According to Figure R2, for the NaOH-treated samples, which dissolve silica and measure total amounts of silicon, yield an extremely significant ANOVA result (p < 0.0001), and the t-tests confirm that one or more silicatein constructs produce a highly significant increase in absorbance compared to the control. In contrast, according to Figure R3, the significant ANOVA result (p < 0.01) for the non-NaOH group indicates that there is a statistically detectable variation in the soluble silicon background levels across all samples. However, the t-tests, which compare each sample directly to the control, all result in non-significant, indicating without the dissolution of NaOH, samples do not have higher concentrations of soluble silicon compounds than the control. This validates that the experimental conditions are well-controlled and that any strong signal observed in the previous NaOH-treated group must come from a different source—which is the solid silica that NaOH dissolved into a detectable form.
According to Figure R4, the test was successful because the control groups showed significantly lower amounts of expected biosilica. The experimental groups were able to catalyze considerable amounts of silica and all plasmids showed stability in silica production. The formation ability among the three experimental groups did not show significant differences, indicating that they could all perform well in silica production. An ANOVA statistical test was performed (p < 0.01). This result meant that the differences in biosilica mass observed between the groups were statistically significant and not due to random chance, further confirming the success in achieving steady and reliable silica formation. The asterisks on each sample signified that the expressed silicatein produced a mass of biosilica that was highly significantly greater than that of the control group in a t-test.
Conclusion:
The addition of other functional proteins—namely, csgA, INP, and YFP—to the plasmid [1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA did not impair its silica-forming activity. In the case of [2] pET-J23119-INP-silicatein, even though the RBS was removed, no decline in silica-forming activity was observed. These results indicated that our new constructs catalyzed a similar amount of silica as [8] pET-T7-RBS-INP-silicatein (K1890001)-T7 tag did, confirming the viability of our plasmid designs. Therefore, the design was successful. For [1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, it yielded a plasmid with expanded functionality while maintaining stable and effective silica formation.. For [2] pET-J23119-INP-silicatein, it demonstrated that even without the RBS, the ability of silicatein to catalyze silica was not reduced. These findings suggested that the RBS could be omitted in future designs.
This test successfully confirmed the ability of our engineered plasmids to catalyze biosilica formation from the precursor TEOS. The data clearly demonstrated that the expressed silicatein was biologically active and capable of driving significant biomineralization. These results supported the proposed application in which engineered bacteria could be injected into building cracks and produce sufficient silica to fill them, thereby fulfilling our primary design objectives.
After testing the successful formation of silica in bacteria culture with the addition of orthosilicate, we tried to test its ability in real-life situations. Since our team went to Professor Yuen’s Laboratory and put some limestones on the hydraulic machinery(See Human Practices), we obtained some limestones with cracks on it. Therefore, we want to test whether our engineered E. Coli forms silica on the surface of limestones with the presence of the csgA adhesion protein.
Silica formation on limestones
Aim:
The silica formation test on limestones is carried out to test if our engineered E. Coli with the csgA protein forms more silica on limestones than the one without.Mechanism:
We observed the cracks on the surface of limestones before doing any experiments. After that, we soaked them into our bacteria with the addition of tetraethyl orthosilicate. Taking the limestones out, we let it dry in air and observe it under the microscope again. If silica is present, the cracks should be narrower after drying.
Protocol:
Preparation:
We shaked and incubated 100mL of bacteria culture ([1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2] pET-J23119-INP-silicatein, [8] pET-T7-RBS-INP-silicatein (K1890001)-T7 tag, and control BL21-pET11a) with LB broth and Amp overnight.
Only for [8] pET-T7-RBS-INP-silicatein (K1890001)-T7 tag: Add IPTG when OD600 = 0.6, incubate and shake at 25C 220rpm overnight.
Testing:
- We observed the cracks on the surface of the limestones with an optical microscope and recorded the position of the cracks we are going to track on.
- Add a final concentration of 4mM tetraethyl orthosilicate to the bacteria culture.
- Soak the limestones in the bacteria horizontally into the bacteria culture ina beaker.
- Place them in the incubator and incubate it for 48 hours without shaking at 37°C.
- After 48 hours, gently take the limestones out of the beaker and put them on a plate each.
- Let it dry at room temperature in air with a cover.
- Fully dried, the limestones is observed under the microscope to see if the cracks have changed.
In Fig. R6, the crack is narrower after being soaked in [1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA bacteria and 4mM tetraethyl orthosilicate for 48 hours. However, the other three, which are [2] pET-J23119-INP-silicatein bacteria(Fig. R7), [8] pET-T7-RBS-INP-silicatein (K1890001)-T7 tag(Fig. R8), and control group BL21-pET11a(Fig. R9), have no difference in the size of their cracks.
Conclusion:
This test verify that our new composite part [1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA does have capability in narrowing cracks on real-life building substrate with biosilica and the addition of adhesion protein csgA, proving that our project has the potential and ability to implement in real-life situations.1. Coradin, T., Eglin, D., & Livage, J. (2004). The silicomolybdic acid spectrophotometric method and its application to silicate/biopolymer interaction studies. Spectroscopy, 18(4), 567–576. https://doi.org/10.1155/2004/356207
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