USAFA - iGEM 2024

Background
Plasmid for INP-sil
Biomineralization IR Spectroscopy
Results and Discussion
Ammonium Test
Ammonium Quantification
Future Directions

Overview

The data presented on this page suggests that E. coli INP-sil effectively facilitates biosilicification and the formation of amorphous aluminosilicate in aluminum-rich environments. Despite concerns about the efficacy of the ammonium test kit for bacterial test samples, the data suggest that E. coli INP-sil produces approximately 27.5x less ammonium than S. pasteurii, the standard bacterium used in biocementation.

Original Project Aims

(a) Verify the production of SiO₂ by E. coli INP-sil and successfully form a biologically-produced aluminosilicate by inducing biosilicification in an environment containing aluminum.
(b) Determine the ammonium concentration produced by S. pasteurii compared to E. coli INP-sil to support the claim that biosilicification is more environmentally friendly than contemporary biocementation.
(c) Upon successful production of an amorphous aluminosilicate by E. coli INP-sil, the aim is to compare the absorptive capabilities of zeolites versus the biologically-produced aluminosilicates in reduction of ammonia byproduct.

Background

In Sporosarcina pasteurii (S. pasteurii), MICP releases a high concentration of ammonia as a byproduct, posing potential problems in the application of biocementation (Carter, 2023). Ammonia is a compound which, when introduced into the environment, can be harmful to many organisms. This becomes a source of concern for real-world applications because of the potential for ammonia-contaminated runoff from biocement products. To effectively apply biocementation, this is a problem that is vital to solve. In the world of wastewater, extensive research has been done on cleaning up ammoniacal water sources (Adam, 2020). One common method has been the application of zeolites --- “aluminosilicate minerals with rigid anionic frameworks containing well-defined channels and cavities” (Pavelic, 2018). Due to these minerals’ ion-exchange properties, they have a great capacity to absorb ammonium, specifically in aqueous solutions. It has been suggested that biological platforms can be harnessed to naturally create aluminosilicates similar to zeolites. Currently, biosilification processes – being those that involve a cell-induced precipitation of silica structures – have been transferred from sponges to bacteria via a silicatein gene. Additionally, research done on diatom biosilification has suggested that the introduction of aluminum into the environment of a silica-producing organism could lead to formation of zeolite-like aluminosilicates (Kohler, 2017). While the biosilification of diatoms differs from that of sponges, it is proposed that introduction of aluminum in the environment of bacteria engineered with silicatein could result in the formation of aluminosilicate with absorption capabilities comparable to zeolites.

Plasmid for INP-sil

Description of INP:

Ice Nucleating Proteins (INPs) are naturally occurring proteins used in several species to facilitate ice formation. INPs naturally migrate to the outer surface of cells. By linking a protein in this case, silicatein-α, INPs act as transporter and have been shown to transport other proteins extracellularly (Kim and Ku, 2018). INP has a long history of successfully transporting enzymes to the outer cell membrane (Jung et al., 1998) making it a promising addition for our team.

Description of silicatein-α:

The silicatein-α gene is an engineered and truncated version of Suberites domunculas silicatein-α gene that has been codon optimized for E. coli. It was engineered by and gifted from collaborators at the University of Virginia. This gene produces the silicatein-α enzyme which acts as a scaffolding site to catalyze the biomineralization of silica spicules (Gao et al. 2024). In a lab setting, this enzyme has catalyzed the formation of calcium carbonate and silicon dioxide spicules (Natalio et al. 2013; Sadeghnejad 2019). Thus, our team has decided to use the silicatein-α gene for the biomineralization of orthosilicate to produce silicon dioxide. We have entered this truncated silicatein-α gene as a new part in the igem registry (BBa_K5199003)

Function of INP-sil:

Contemporary biocementation releases ammonium as a byproduct that leeches into the soil and poses potential environmental risks (Chuo et al. 2020, Esteban et al. 2016). The purpose of our INP and silicatein-α composite part is to function as an ammonium free alternative for biocementation. Silicatein-α serves as the biomineralization site, capable of creating an amorphous mixture of CaCO₃ and SiO₂ without the resulting ammonium byproduct (Natalio et al. 2013). INP functions as an extracellular transporter allowing the silicatein-α enzyme to be expressed on the external surface of E. coli where the biomineralization is occurring (Kim and Ku, 2018). Due to the passive nature of silicatein-α, this allows the enzyme to continue the biomineralization process even when the bacteria are dead, eliminating safety concerns of introducing living genetically modified organisms into the environment (Bawazer, 2013). This provides a safer and environmentally friendly alternative to contemporary biocementation that can be used for a myriad of applications. The INP-sil composite part is BBa_K5199004.

Biomineralization IR Spectroscopy

Design 1:

The objective was to create a biomineralization assay to verify and understand the production of CaCO₃ and SiO₂ under different conditions as well as create an amorphous aluminosilicate. After consulting with Dr. Knoerzer, we determined that IR spectroscopy would be the most efficient method for analyzing mineral production. Key test groups were identified:

Chemical-only controls
S. pasteurii (contemporary biocement control)
E. coli INP-sil (engineered for silica production)
Wild-type E. coli (non-engineered control)

Additionally, based on preliminary literature reviews, a 1:1 ratio of aluminum to silica was used in some tests to stimulate amorphous aluminosilicate formation.

Build 1:

A protocol was developed to carry out the assays for each of the groups.
Using ultracentrifugation, pellets of biological material were prepared for each test group.
Each pellet was extracted and dried overnight for IR.

Test 1:

The IR spectroscopy scans were successfully performed on the pellet samples, but the air drying of samples was ineffective as the pellets were semi-solid with noticeable water present.
For the most part, the results aligned with the expectations, however, the scans were partially masked by excess water present in the samples, which interfered with accurate readings of mineral composition.
The initial test using the 1:1 Al/Si ratio did not match the preliminary literature review, prompting further investigation and suggesting a combination of 2:1 Al/Si was formed.

Learn 1:

Water interference in the IR scans showed that the samples needed to be properly dehydrated before IR analysis to prevent masking of the mineral signals.
Further literature review suggested a 2:1 Al/Si ratio was more appropriate for forming amorphous aluminosilicate, which was different from the 1:1 ratio used initially.
Adjustments to the assay conditions, including both the dehydration process and the Al/Si ratio, were identified for the next round of testing.

Design 2:

The objective remained to verify and understand the production of CaCO₃, SiO₂, and amorphous aluminosilicate. After addressing water interference issues in IR spectroscopy, the focus shifted to refining the assay with better dehydration techniques and testing for brick matrix formation using combined silicon dioxide and calcium carbonate production. Key groups tested:

Chemical-only controls
S. pasteurii (contemporary biocement control)
E. coli INP-sil (engineered for silica production)
Wild-type E. coli (non-engineered control)

Build 2:

Dehydration process: The samples were air-dried for a week on absorbent towels with the purpose of reducing water interference.
Assay protocol revisions incorporated changes based on literature to test for 2:1 Al/Si ratios for amorphous aluminosilicate.
Assays for the chemical controls were expanded to include soda lime to create conditions more similar to contemporary cementation.

Test 2:

IR spectroscopy on the air-dried pellets showed significant improvements, as water interference was reduced.
E. coli INP-sil samples produced noticably larger pellets than WT E. coli and chemical controls. Unfortunately, both WT E. coli pellets suspended in water dissolved into solution upon extraction preventing IR data from being recorded for these samples.
Strong signals for SiO₂ bonds were detected in the E. coli INP-sil group, though WT E. coli and chemical controls also showed silicon dioxide production.
No calcium carbonate production was seen in E. coli INP-sil in urea/calcium solution, correlating with brick data showing that 100% TB-Si solution outperformed the urea/calcium solution.
Evidence of amorphous aluminosilicate formation was observed when aluminum hydroxide was added and a larger pellet was formed when using the 2:1 Al/Si ratios compared to the previous 1:1 test ratio.
Chemical controls using soda lime showed the presence of both CaCO₃ and SiO₂, offering another potential avenue for brick matrix optimization.

Learn 2:

Although spontaneous mineralization was observed, the larger pellet sizes of E. coli INP-sil samples compared to WT E. coli and chemical controls suggests the effective facilitation of silica polymerization by E. coli INP-sil.
Proper dehydration was essential for eliminating water interference and improving accuracy in IR spectroscopy.
The presence of SiO₂ in multiple groups showed that while INP-sil guided the process, it was not the only contributor to silica formation.
The absence of calcium carbonate in E. coli INP-sil in urea/calcium solution provided further insight into brick formation, requiring further exploration to create a combination of SiO₂ and calcium carbonate in the brick matrix.
Soda lime provided an additional method for generating calcium carbonate, hinting at its potential role in future testing of mixed brick matrices.

Results and Discussion:

After centrifuging the E. coli in Terrific Broth (TB), the supernatant was removed and replaced with water, showing that the cells could still polymerize in a water environment. Both E. coli INP-sil and E. coli wild type (WT) cells were resuspended in water along with various chemicals, following the growth and protein induction phases. Remarkably, the cells were able to perform biomineralization in water, and without the presence of TB. This explains why the plant and dust groups switched to water-Si instead of TB-Si, aiming to reduce possible ammonium content and prevent plant damage.

_{Figure 1. IR of E. coli INP-sil, E. coli INP-sil and aluminum, and Calcium Carbonate}

In the first iteration of the Infrared (IR) Spectroscopy testing, the pellets in Figure 1 were run and resulted in the same peaks/oscillations. Figure 1 does not match Figure 2, 3, or 4 standard IR spectrums, concluding that the first iteration of testing the pellets was overconcentrated with water which diluted the pellet to express H₂O peaks. To overcome this issue, in the second iteration the pellets were spun down more to be more compact and then dried out to keep the water out of the IR spectrums. The results of the second iteration of testing are in the IR spectrum Figures below. The analysis of 1b, 2b, and 3b was carried out using IR spectroscopy. We completed these IR testing iterations and analyzed the spectrums with the aid of Dr. Knoerzer.

_{Scheme 1. Condensation of Two Orthosilicate Molecules with the Elimination of Water to form 1b.}

_{Scheme 2. Equilibrium equations of Calcium Carbonate, 2b.}

_{Scheme 3. Transformation of Aluminum Silicate, 3b.}

_{Figure 2. IR of Silicon Dioxide (pure sample)}

For IR analysis as shown in Figure 2 from the Synthesis of amorphous silica and sulfonic acid functionalized silica used as reinforced phase for polymer electrolyte membrane, only product 1b is present (Tran). There is an alcohol group at a broad peak around 3400cm^-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. There is a minor peak around 1630cm^-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peaks from around 1100cm^-1 to 500cm^-1 signal the presence of Si-O-Si alone. All these peaks are indicative of a pure silicon dioxide product (1b).

_{Figure 3. IR of Calcium Carbonate (pure sample)}

For IR analysis as shown in Figure 3 from the National Institute of Standards and Technology, only the product 2b is present (NIST). The most prevalent functional group is carbonate (CO₃^2-). It is shown with a sharp peak around 1500cm^-1. In 2b, there is another strong peak around 1000cm^-1, which displays a carboxylic acid OH bond on bicarbonate (HCO₃^-). These peaks are indicative of 2b, a pure calcium carbonate product.

_{Figure 4. IR of Amorphous Hydrated Aluminum Silicate}

For IR analysis as shown in Figure 4 from the Kinetic study of removal heavy metal from aqueous solution using the synthetic aluminum silicate, only product 3b is present (Treto-Suarez). There is an alcohol group at a broad peak around 3400cm^-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. There is a minor peak around 1630cm^-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peak from around 1100cm^-1 signals the presence of Si-O-Si alone. Around the 500 to 1500cm^-1 range shows the different types of peaks within the structures O−Si−O and Al−O−Si (Treto-Suarez). The most notable peak in this range is Al-O-Si around 500 to 1000cm^-1. There are two peaks around 1630cm^-1 to 1330cm^-1, which is attributed to water within the Si-O-Si structure (Ellerbrock). These peaks are indicative of an amorphous hydrated aluminum silicate (3b).

_{Figure 5. IR of Chemical Control with Soda Lime}

For IR analysis as shown in Figure 5, it displays the product 1b and 2b. There is an alcohol group at a broad peak around 3400cm^-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. The peak is shown by a Si-O-H annotated on the spectra. There is a minor peak around 1630cm^-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peaks from around 1100cm^-1 to 500cm^-1 signal the presence of Si-O-Si alone. All these peaks are indicative of Figure 2, which is a pure silicon dioxide product (1b). Additionally, the product 2b is also present in Figure 4, with the most prevalent functional group carbonate (CO₃^2-). It is shown with a sharp peak around 1500cm^-1. In 2b, there is another strong peak around 1000cm^-1, which displays a carboxylic acid OH bond on bicarbonate (HCO₃^-). These peaks are indicative of 2b, a pure calcium carbonate product. Figure 4 includes both the silicon dioxide and calcium carbonate as products on the IR spectra.

_{Figure 6. IR of E. coli INP-sil}

For IR analysis as shown in Figure 6, only product 1b is present. There is an alcohol group at a broad peak around 3400cm^-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. The peak is shown by a Si-O-H annotated on the spectra. There is a minor peak around 1630cm^-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peaks from around 1100cm^-1 to 500cm^-1 signal the presence of Si-O-Si alone. All these peaks are indicative of Figure 2, which is a pure silicon dioxide product (1b).

_{Figure 7. IR of Chemical Control without Soda Lime}

For IR analysis as shown in Figure 7, only product 1b is present. There is an alcohol group at a broad peak around 3400cm^-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. The peak is shown by a Si-O-H annotated on the spectra. There is a minor peak around 1630cm^-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peaks from around 1100cm^-1 to 500cm^-1 signal the presence of Si-O-Si alone. All these peaks are indicative of Figure 2, which is a pure silicon dioxide product (1b).

_{Figure 8. IR of Wild Type E. coli (Cement Solution)}

For IR analysis as shown in Figure 8, only product 1b is present. There is an alcohol group at a broad peak around 3400cm^-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. The peak is shown by a Si-O-H annotated on the spectra. There is a minor peak around 1630cm^-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peaks from around 1100cm^-1 to 500cm^-1 signal the presence of Si-O-Si alone. All these peaks are indicative of Figure 2, which is a pure silicon dioxide product (1b).

_{Figure 9. IR of E. coli INP (Cement Solution)}

For IR analysis as shown in Figure 9, only product 1b is present. There is an alcohol group at a broad peak around 3400cm^-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. The peak is shown by a Si-O-H annotated on the spectra. There is a minor peak around 1630cm^-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peaks from around 1100cm^-1 to 500cm^-1 signal the presence of Si-O-Si alone. All these peaks are indicative of Figure 2, which is a pure silicon dioxide product (1b).

_{Figure 10. IR of S. pasteurii}

For IR analysis as shown in Figure 10, only product 2b is present. The most prevalent functional group is carbonate (CO₃^2-). It is shown with a sharp peak around 1500cm^-1. In 2b, there is another strong peak around 1000cm^-1, which displays a carboxylic acid OH bond on bicarbonate (HCO₃^-). These peaks are indicative of Figure 3, a pure calcium carbonate product (2b).

_{Figure 11. IR of E. coli INP-sil and aluminum}

For IR analysis as shown in Figure 11 from the Kinetic study of removal heavy metal from aqueous solution using the synthetic aluminum silicate, only product 3b is present (Treto-Suarez). There is an alcohol group at a broad peak around 3400cm^-1. This alcohol group is coupled with silicon, making the alcohol more diluted and not as strong of a peak. There is a minor peak around 1630cm^-1, which is water within the Si-O-Si structure (Ellerbrock). Additionally, the peak from around 1100cm^-1 signals the presence of Si-O-Si alone. Around the 500 to 1500cm^-1 range shows the different types of peaks within the structures O−Si−O and Al−O−Si (Treto-Suarez). The most notable peak in this range is Al-O-Si around 500 to 1000cm^-1. There are two peaks around 1630cm^-1 to 1330cm^-1, which is attributed to water within the Si-O-Si structure (Ellerbrock). These peaks are indicative of an amorphous hydrated aluminum silicate (3b).

Ammonium Test

Design 1:

The goal was to measure the initial ammonium concentrations in flowthrough samples from bricks made with:

S. pasteurii (contemporary biocement control)
E. coli INP-sil (engineered for silica production)

This would allow us to assess potential environmental concerns by comparing the ammonium production of these different bacteria and understand how varying test conditions influence ammonium concentrations. We decided to use an ammonium test kit utilizing an enzyme coupled reaction and a plate reader for quantifying ammonium levels.

Build 1:

The protocol involved creating bricks from each bacterial strain, then collecting flowthrough samples from these bricks.
Ammonium concentrations in these samples were measured using the ammonium test kit, which involved preparing standards and samples to generate a standard curve for accurate quantification.
Three different individuals followed the protocol over three separate test runs to ensure repeatability and eliminate user error as a factor.

Test 1:

All three rounds of testing failed to produce a usable standard curve, meaning that the concentration readings were unreliable and could not be accurately interpreted.
The test results indicated possible issues with either the ammonium test kit, the protocol associated with the kit, or the plate reader.
Despite following the correct procedure, there was no consistency in the readings, and the standard curve was erratic, preventing any conclusions from being drawn about ammonium levels.

Learn 1:

The failure to generate a usable standard curve in all three trials strongly suggests that there might be a defect in the test kit, the protocol, or an issue with the plate reader. Potential sources of the problem could include:

Expired or degraded reagents in the ammonium test kit. Particularly the enzyme used for the reaction.
Malfunctioning plate reader or improper calibration, resulting in inaccurate absorbance readings.
Aerosolization of nearby wells leading to disturbances in absorption.

Design 2:

The goal was to measure ammonium concentrations in flowthrough samples from bricks made with:

S. pasteurii (contemporary biocement control)
E. coli INP-sil (engineered for silica production)

This would help assess potential environmental concerns by comparing ammonium production between these bacteria. An ammonium test kit with an enzyme-coupled reaction and a plate reader was selected for quantification.

Build 2:

The protocol involved creating bricks from each bacterial strain, then collecting flowthrough samples from these bricks under various conditions.
Ammonium concentrations in these samples were measured using the ammonium test kit, which involved preparing standards and samples to generate a standard curve for accurate quantification.
To address concerns about aerosolization affecting the concentrations, samples were spread out across the plate.

Test 2:

Spreading out samples on the plate helped resolve aerosolization issues. A standard curve was generated (R² = 0.9998), confirming the test kit's ability to accurately quantify standard ammonium concentrations.
However, serial dilutions of E. coli INP-sil and S. pasteurii samples showed erratic results, suggesting the test kit might not be effective for these specific samples. The dilution data did not follow expected trends, and the readings were inconsistent.
The erratic results point to potential interference from the samples, possibly due to pH differences affecting enzyme activity or biological compounds from the bacteria inhibiting enzyme function.
Despite inconsistencies, the data suggested that E. coli INP-sil produced substantially less ammonium than S. pasteurii, though not completely ammonium-free as initially hypothesized.

Learn 2:

The spread-out plate layout helped eliminate aerosolization as an issue, confirming that previous erratic results were likely due to improper sample handling.
While the standard curve demonstrated that the test kit worked well for known ammonium concentrations, the erratic results for the biological samples suggest potential limitations in the kit’s applicability to samples with varying pH or biological interference.
The hypothesis that E. coli INP-sil would be ammonium-free was only partially supported; it produced substantially less ammonium than S. pasteurii but not none, raising new questions about its role in biocementation.
Future tests need to account for sample pH and potential biological interference with the enzyme-coupled reaction. A buffer or pretreatment step might be necessary to stabilize the pH or remove inhibitory compounds. Further investigation into these variables will improve the reliability of ammonium quantification.

Ammonium Quantification:

_{Figure 12. Standard curve of ammonium dilutions measured at 660 nm for determining ammonium concentration in test samples. Displays a standard curve with an R² of 0.9998 and a line of y=1321.3x.}

_{Figure 13. Graph showing the predicted ammonium concentration of five S. pasteurii dilutions calculated using the OD at 660 nm and the standard curve equation found in Figure 12.}

_{Figure 14. Graph showing the predicted ammonium concentration of five E. coli INP-sil dilutions calculated using the OD at 660 nm and the standard curve equation found in Figure 12.}

As shown in Figures 13 and 14, the predicted ammonium concentrations for each dilution of the S. pasteurii and E. coli INP-sil dilutions was not consistent and showed some considerable variability. Ideally, each dilution would have an equal ammonium concentration when using the optical density in the standard curve and correcting for the dilution factor to determine the equivalent concentration of the undiluted test sample. The inconsistencies present in both the S. pasteurii and E. coli INP-sil dilutions suggests that the ammonium test kit used may not be an effective test method for our samples. Because the ammonium test kit relies on an enzyme coupled reaction for the colorimetric changes to occur, it is possible that the pH of the test samples is altering enzyme activity, the bacteria in the test samples produces a compound leading to partial inhibition of the enzymes, or the presence of bacteria in the test samples is leading to enzyme degradation in some way.

_{Figure 15. Graph comparing the average calculated ammonium concentration for S. pasteurii and E. coli INP-sil dilutions with an OD within the standard curve.}

Although the data in Figures 13 and 14 suggests that the ammonium test kit provides erratic results when applied to bacterial test samples, the ammonium concentrations for average dilutions of S. pasteurii and E. coli INP-sil were compared in Figure 15. Using only dilutions with an optical density within the standard curve, an average ammonium concentration was calculated for the predicted undiluted test samples. These results suggest that there is approximately 27.5x as much ammonium produced by S. pasteurii than E. coli INP-sil. This matches our initial hypothesis because contemporary biocementation with S. pasteurii relies on the enzyme urease to break down urea and ultimately generates the production of ammonium ions. This enzyme is not present in E. coli INP-sil and helps explain the disparity between ammonium production.

Future Directions

Given the successful production of amorphous aluminosilicate as suggested by our IR data, a key next step will be to incorporate this material into brick production. We aim to assess how the inclusion of amorphous aluminosilicate affects both ammonium concentration in flowthrough samples and the overall strength of the bricks. This will help us understand its potential as a biocompatible and environmentally friendly material for construction applications. Additionally, we plan to test the effectiveness of zeolite in the brick matrix to determine its ability to absorb ammonium ions. Zeolites are known for their ion-exchange properties, and their inclusion in the flowthrough system may offer a novel way to mitigate ammonium release from biocementation processes, addressing environmental concerns associated with high ammonium concentrations. By comparing the ammonium levels and mechanical properties of bricks containing amorphous aluminosilicate, zeolite, or a combination of both, we will be able to optimize the material composition for environmental sustainability and structural integrity. These future tests will provide critical data for developing more sustainable, ammonium-reducing alternatives to traditional biocementation, enhancing both ecological impact and material performance.

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