1. Aim:

The goal of this test is to confirm the viability of three plasmids—[1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2] pET-J23119-INP-silicatein, and [8] pET-T7-RBS-silicatein (K1890001)-T7 tag—for expressing silicatein. We aim to determine whether the expressed silicatein can successfully catalyze the formation of biosilica and to quantify its production, thereby validating our plasmid design.

2. Design:

Silicatein facilitates the production of silica from monomeric silicon compounds. We tried to search for tests that could visualize and quantify the production of silica. However, due to its chemical inertness [2], silica do not easily react with most substances. We decided to use the silicomolybdate blue method to determine the concentration of silicon from a calibration curve between known concentrations and their corresponding OD values. The larger the OD value, the higher the concentration of silicon [7].

Silica is first transformed into a reactive form, then ammonium molybdate is added to produce Mo+6 ion, which is then reduced by ascorbic acid to form Mo+5 ion, showing blue color formation [1] [6] [9]. Oxalic acid serves as a masking agent to suppress interference from phosphate, since it also reacts with ammonium molybdate and forms a similar blue color [7]. Glycerin is added to act as a stabilizer. By acquiring the concentrations of Si, we could further calculate the estimated mass of SiO2 and quantify its production.

Other researchers also adopted this method to determine silicon in silica samples [7] [5]. Tetraethyl orthosilicate (TEOS), a liquid form of silicon compound at room temperature, is used as the precursor for silicatein [8].

For expected data, the control group should show very low OD values under spectrophotometer scan compared to experimental groups since it does not express silicatein. Therefore, silica quantification for the control group is also expected to be significantly lower than that of experimental groups. The control group cannot synthesize biosilica.

Key points:

• Ascorbic acid is the reducing agent.
• Oxalic acid, a masking agent, prevents interference of phosphate compounds.
• Glycerin as stabilizer.

3.1.1 Build:

In the first test, all groups of bacteria (BL21-pET-11a, [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag) were treated with 4mM TEOS. After incubation, the samples were centrifuged and the precipitates, which were the cell pellets, were removed. The supernatants were resuspended in water and underwent a second round of centrifugation. Then, the supernatants were removed, and ideally, the precipitates at the bottom of the incubation tubes were the biosilica [10]. No silica precipitate could be observed by the naked eye due to small quantities. NaOH was directly added to the samples to break down silica polymers and HCl was added to adjust the pH [5] [10]. Acidified ammonium molybdate, oxalic acid, ascorbic acid, and glycerin solution were added one after another. The samples were left for blue color formation, before being scanned under 825 nm to determine their OD values. The test was conducted twice.

Key points:

• TEOS is a liquid compound, it should be removed from samples by centrifugation and not interfere with the test results. Thus, silicon in the remaining samples should all come from silica.
• NaOH is used to transform unreative silica into reactive silica. Only when silica is dissolved into silicic acid or silicates can silicon be released from the polymer.

3.1.2 Tests/Results:

The experiment yielded statistically non-significant results. The ANOVA test for both the OD values, which correlate with soluble silicon concentration, and the direct mass quantification of biosilica showed no significant differences across the samples. The individual t-test results when compared to the control confirm that none of the engineered plasmids enhanced silica production. The first test also failed due to larger amounts of biosilica produced by the control group, which was abnormal since it did not express silicatein.

3.1.3 Learn:

After discussing with professors and teammates, we suspected that phosphate in the cells could react with ammonium molybdate and interfere with the results. However, oxalic acid had been added to the protocol to prevent this. We also considered reagent contamination as a source of silicon, but this was ruled out when a control experiment using only DDH₂O yielded no blue color. Other research indicated the use of HF instead of NaOH [7]. Thus, we decided to adopt HF in the next experiment to check whether it could better break down silica polymer than NaOH did. We suspected that NaOH might not be competent enough to dissolve all silica, thus the test results failed to manifest the total amounts of biosilica produced.

3.2.1 Rebuild:

In the second test, all procedures remained the same, but instead of adding NaOH and HCl, 5% HF was added to each sample. We expected that by changing the method to dissolve silica, more silica could be broken down and react with the reagents, yielding more ideal results, that is, higher OD values and biosilica mass in all experimental groups than in the control. The test was only conducted once due to time constraint.

Key points:

• HF, instead of NaOH, was used to check wheter it could transform more silica into reactive forms in this test.

3.2.2 Tests/Results:

Even though the method used to dissolve the silica monomer was changed, according to Figure 3, the abnormal blue color formation in the control groups was still present, indicating that the experiments still contained problems. Besides, all OD values decreased significantly from the previous test.

3.2.3 Learn:

After reviewing our referring literature [7], we found that silica must be first transformed into an amorphous form before reacting with HF. It was possible that the neglect and ignoring of this transformation resulted in the abnormality. We learnt that experimental procedures needed to be analyzed and double-checked in a more detailed and careful manner before future tests were conducted to avoid such incidence. However, since the steps indicating the transformation of silica were unclear and ambiguous, we then searched for other literature adopting the same HF method. We failed because most research used NaOH instead of HF, thus we decided to use NaOH again next time.

3.3.1 Rebuild:

In the third test, all groups of bacteria (BL21-pET-11a, [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag) were treated with 4mM TEOS. This test followed the procedure of the NaOH-HCl method.

However, samples underwent a sonication process to extract protein (silicatein) and directly react with TEOS. This was inspired by the procedures of Western Blot [4]. In Western Blot, bacterial samples were centrifuged to extract proteins and test protein levels. We suspected that other components in cultured bacteria might react with TEOS and interfere with the results, so we devised a method to directly test the effectiveness of silicatein to catalyze silica. The test was more targeting and only focused on the activeness of silicatein. Moreover, this test introduced a new method to further rule out the effect of other silicon content on the experimental results. We suspected that centrifugation would not be an effective method to exclude TEOS since the method failed in the first two tests. Some of which might stick to the silica samples and interfere with the results.

After incubation, the samples were centrifuged and the supernatants were removed. The precipitates were resuspended in PBS solution and sonicated with 75 kHz. After sonication, samples were centrifuged and the cell debris was removed. The supernatants contained the silicatein expressed by the bacteria. TEOS was then added to the supernatants, and samples were incubated overnight. After incubation, the samples were centrifuged and the precipitates were acquired. They were resuspended in water, and some portions of which were removed to directly react with acidified ammonium molybdate, oxalic acid, ascorbic acid, and glycerin solution. This group of samples was named the non-NaOH samples. NaOH and HCl were added to the remaining samples, and other reagents were also added. This group of samples was named the NaOH-treated samples.

The optical density of both the NaOH-treated and non-treated groups was measured to determine their corresponding silicon concentrations. The NaOH-treated group quantified the total silicon content of the original sample, while the non-NaOH group represented the silicon from all sources except solid silica, which remained undissolved. The corresponding silicon masses for the original sample were calculated from these concentrations. The mass from the non-NaOH group was subtracted from that of the NaOH-treated group to isolate the mass of silicon derived exclusively from the dissolved, catalyzed silica. This differential calculation corrects for interference from residual TEOS and other soluble silicon compounds. The resulting purified silicon mass was then multiplied by the stoichiometric factor of 2.143 to report the final mass as silicon dioxide (SiO₂). The entire assay was performed twice.

Key points:

• Centrifugation may not be sufficiently effective to remove irrelevant silicon sources from samples, such as residual TEOS.
• Sonication was introduced to extract silicatein and directly test its activity and viability.
• "Mass subtraction" method was introduced to exclude irrelevant silicon sources from interferring test results.

3.3.2 Tests/Results:

The third test was successful because it was the first time the control groups showed the lowest precipitate mass. The subtraction of the silicon mass in the NaOH-treated group from the non-NaOH group helped provide a more accurate and ideal experimental result, successfully excluding the effects of remaining TEOS and other irrelevant silicon compounds. However, the direct mass measurement of biosilica showed no significant difference from the control. This indicated that while the plasmids successfully expressed active silicatein, the results were not significant and relatively random. The amount of biosilica formed in BL21-pET-11a was still not significantly different from the experimental groups.

3.3.3 Learn:

Overall, this test was relatively successful since expected patterns could be observed. We considered that after adding NaOH, the samples should be heated to ensure complete dissolution of the silica [3], facilitating a complete manifestation of silica formation.

3.4.1 Rebuild:

In the fourth test, all the procedures were the same as the third test. However, sonication was not performed due to a limited amount of time. NaOH-treated samples were heated using a water bath before other reagents were added.

Key points:

• Samples were heated after NaOH was added to facilitate complete transformation from unreactive to reactive silica

3.4.2 Results:

3.4.3 Conclusion:

The last experiment was successful due to the extremely low precipitate mass in BL21-pET-11a and a significant mass difference between the experimental groups and the control group (according to t-test results in Figure 10). The results were not random, which was proven by ANOVA. The data collected were ideal, indicating that our designed plasmids [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA and [2]pET-J23119-INP-silicatein were capable of expressing silicatein and producing silica. This experiment indicated that sonication might not be an essential step since its removal did not negatively affect the test results. However, introducing the NaOH-treated and non-NaOH samples was essential since it helped exclude other silicon content affecting the test results, thus providing more accurate, ideal, and expected test results which only represented the amounts of silica formed.

Through cycles of trial and error, our team succeefully perform the silicomolybdate blue assay and confirm the viability of our plasmids. We rebuild and adjust our method for multiple times in order to yield the best results representing the success of our plasmid designs

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