iGEM 2025 — Team puiching-macau

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## Overview

During our iGEM year, we organized our project by performing wet lab experiments.

Weeks

* Protocols
* Week 1 (May 18 - May 24)
* Week 2 (May 25 - May 31)
* Week 3 (June 1 - June 7)
* Week 4 (June 8 - June 14)
* Week 5 (June 15 - June 21)
* Week 6 (June 22 - June 28)
* Week 7 (June 29 - July 5)
* Week 8 (July 6 - July 12)
* Week 9 (July 13 - July 19)
* Week 10 (July 20 - July 27)
* Week 11 (July 28 - August 3)
* Week 12 (August 4 - August 10)
* Week 13 (August 11 - August 17)
* Week 14 (August 18 - August 24)
* Week 15 (August 25 - August 31)
* Week 16 (September 1 - September 7)
* Week 17 (September 8 - September 14)
* Week 18 (September 15 - September 21)

#### Protocol for tests

---

#### Week 1

---

**1. Brainstorm & Design of Plasmids**
Date: 16/05/2025
Aim: Brainstorm & design plasmids for our project before material preparation and subsequent experiments.
![](https://static.igem.wiki/teams/5685/maynotebook/brainstorm.webp)

Figure 1.1. Our team members brainstorming plasmid designs

**1. Preparation of LB broth and LB agar**
Date: 19/05/2025
Aim: Provide essential nutrients for the rapid growth and maintenance of E. coli and other bacteria.

Protocol:
Measure 36g of LB agar powder and 1000mL of MiniQ water. Add 36g LB agar powder to 1000mL MiniQ and mix thoroughly.
Measure 21g of LB agar powder and 1000mL of MiniQ water. Add 21g LB agar powder to 1000mL MiniQ and mix thoroughly.
Results:
LB broth and LB agar were successfully prepared.
**2. Preparation of ampicillin, LB Agar gel plate**
Date: 20/05/2025
Aim: To prepare LB agar plates, both with and without the antibiotic ampicillin, for the growth and selection of transformed E. coli BL21 cells.

Protocol:
Ampicillin stock solution (100 mg/mL) was prepared by dissolving 0.1 g of ampicillin sodium salt in 1 mL of sterile Milli-Q water. For LB agar plates without antibiotic, 200 mL of dissolved agar was cooled to approximately 50°C and poured into Petri dishes. For the antibiotic-containing plates, 200 µL of ampicillin stock was added to 200 mL of cooled dissolved agar (1:1000 ratio, final concentration 100 µg/mL), mixed gently, and poured into plates. All plates were solidified at room temperature. For bacterial streaking, a sterile wire loop was used to collect E. coli BL21 from a stock culture. The sample was streaked onto an LB agar plate and incubated at 37°C overnight.
Results:
100 mg/mL Ampicillin stock, LB Agar gel plate(both with and without the antibiotic ampicillin) were successfully prepared

![](https://static.igem.wiki/teams/5685/maynotebook/20250927233302-95-42.webp)

Figure 1.2. Adding ampicillin into the LB agar plates

![](https://static.igem.wiki/teams/5685/maynotebook/20250928000006-116-91.webp)

Figure 1.3. Transforming LB agar gel to the plate

**3. Competent cell**
Date: 22/05/2025
Aim: To prepare and preserve competent E. coli BL21 cells for use in genetic transformation experiments, ensuring high efficiency for plasmid uptake.

Protocol:
5 mL of cultured BL21 was added into 500 mL of LB broth and it was incubated at 37°C with shaking (220 rpm) until the OD600 value reached between 0.55 and 0.6 (~4.5 hours). The culture was divided into two pre-chilled 250 mL centrifuge tubes and centrifuged (4,000 × g, 5 min, 4°C). The supernatant was discarded, and each pellet was gently resuspended in 10 mL of ice-cold 100 mM CaCl2. After 30 minutes on ice, cells were repelleted by centrifugation and resuspended in 10 mL of ice-cold CaCl2 with 15% glycerol. The suspension was aliquoted (100 µL per tube), flash-frozen in liquid nitrogen, and stored at −80°C.
Results:
Competent E. coli BL21 cells were successfully prepared. All aliquots were flash-frozen and stored at −80°C, yielding transformation-ready competent cells.
**4. Transformation of plasmids**
Date: 23/05/2025
Aim: To introduce plasmids into E. coli BL21 and DH5α strains via heat-shock transformation, enabling plasmid amplification, protein expression.
List of plasmids being transformed:
[1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA
[2]pET-J23119-INP-silicatein
[3]pET-T7-INP-YFP(K523013)-csgA
[6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta
[7]pET-T7-INP-YFP (K523013)-T7 tag
[8]pET-T7-RBS-silicatein (K1890001)-T7 tag

Protocol:
Plasmid DNA was transformed into competent BL21 cells using a standard heat-shock protocol. 1µL of plasmid was gently mixed with cells and incubated on ice for 20 minutes. Cells were heat-shocked at 42°C for 1 minute, returned to ice for 2 minutes, and then recovered in 900 µL LB broth at 37°C for 1 hour with shaking (300 rpm). For plating, 100 µL of the mixture was spread onto each of four LB agar plates containing the appropriate antibiotic. The remaining culture was concentrated by centrifugation (700 µL supernatant removed) and 100 µL of the 4.5× concentrated cells was plated. All plates were incubated overnight at 37°C and stored at 4°C.

Results:
BL21 Transformations: Plasmids [1]-[3] and [6]-[8] were successfully transformed into BL21 cells, as confirmed by colony growth on selective plates.
DH5α Transformations: Plasmids [7] and [8] were successfully transformed into DH5α cells, but transformation of plasmid [6] into DH5α failed (no colonies observed).
Conclusion:
Plasmids [1]-[3] & [7]-[8] were all successful in one run, except for plasmid [6], which facilitated their use in subsequent experiments.

#### Week 2

---

**1. Preparation of Midi of [1]-[8]**
Date: 27/05/2025 & 30/05/2025
Aim: Isolate and purify small amounts of plasmid DNA from bacterial cultures.
List of plasmids being midi prep:

* [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csg
* [2]pET-J23119-INP-silicatein
* [3]pET-T7-INP-YFP(K523013)-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-silicatein (K1890001)-T7 tag

Protocol:
A 100 mL bacterial culture was grown overnight and harvested by centrifugation. The pellet was resuspended, lysed with an alkaline buffer, and neutralized to precipitate genomic DNA and cellular debris. The lysate was clarified by gravity-flow filtration and applied to a silica column. After washing, plasmid DNA was eluted, precipitated with isopropanol, and washed with ethanol. The purified DNA pellet was resuspended in water, and its concentration was quantified by Nanodrop.
Results:
Nanodrop analysis showed a plasmid[1]-[8] from bacterial cultures concentration,plasmid[2]-[6]&[8]were successful. [1],[7]was failed. [2]-[6]&[8]exhibit high concentrations (ranging from ~152 ng/μl to ~341 ng/μl) and excellent purity. Their A260/A280 ratios are very close to the ideal value of 1.8 (all between 1.87-1.93).

Table 2.1. The concentrations and purity of the obtained plasmids

![](https://static.igem.wiki/teams/5685/maynotebook/midiprep-table1.webp)

Conclusion:
plasmids Midi of [2]-[6] & [8] were successful, verifying that the bacterial clones contained the correct plasmids with our desired inserts.

![](https://static.igem.wiki/teams/5685/maynotebook/figure2.webp)

Figure 2.1. Washing components of the nucleobond xtra midi columns

![](https://static.igem.wiki/teams/5685/maynotebook/img-7336.webp)

Figure 2.2. Running plasmids [1][7][8]Midi results

#### Week 3

---

**1. Transformation of plasmid**
Date: 03/06/2025

Aim: Transform [2]pET-J23119-INP-silicatein, [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM into BL21 competent cells.

Protocol:
Transformation was performed using a heat-shock method (20 minutes on ice, 1 minute in a thermoblock at 42°C, and 2 minutes on ice) into competent BL21 cells. The bacteria were streaked onto LB-agar plates and incubated overnight at 37°C. Those not immediately used were stored at 4°C.

Result:
Transformation of all plasmids was successful.

Conclusion:
[2]pET-J23119-INP-silicatein , [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM into BL21 competent cells were all successful in one run.

**2. Preparation of Midi**
Date: 04/06/2025

Aim: To extract and purify the sequences of [1]pET-J23119 INP-silicatein + INP-YFP-csgA, and [7]pET-T7-INP-YFP (K523013)-T7 tag in order to test their concentration.

Protocol:
The procedure isolated plasmid DNA from a 100 mL bacterial culture. After the cells were pelleted by centrifugation (15 min, 4500 × \*g\*), they were resuspended, lysed with Buffer LYS for 4.5 minutes, and neutralized. The resulting lysate was cleared by gravity flow through a pre-equilibrated filter column. The column was washed, and the DNA was eluted with 5 mL of Buffer ELU. The DNA was then precipitated by adding isopropanol and centrifuging (20 min, 4500 × \*g\*). The pellet was washed with 2 mL of 70% ethanol, dried, and finally resuspended in 80 µL of water for quantification via Nanodrop.

Results:
Nanodrop analysis shows concentrations of [1]pET-J23119 INP-silicatein + INP-YFP-csgA, and [7]pET-T7-INP-YFP (K523013)-T7 tag. [7]pET-T7-INP-YFP (K523013)-T7 tag was successful due to high concentration and ideal purity while [1]pET-J23119 INP-silicatein + INP-YFP-csgA failed.

Table 3.1. The concentrations and purity of the obtained plasmids

![](https://static.igem.wiki/teams/5685/maynotebook/table2.webp)

Conclusion:
Midi of [7]pET-T7-INP-YFP (K523013)-T7 tag was successful, verifying that the bacterial clones contain the correct plasmids with our desired inserts. However, [1]pET-J23119 INP-silicatein + INP-YFP-csgA needed further tests due to extremely and abnormally low concentration. Errors and contaminations needed to be avoided.

**3. Transformation of plasmid**
Date: 04/06/2025

Aim:
Transform [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta, [7]pET-T7-INP-YFP (K523013)-T7 tag, [8]pET-T7-RBS-INP-silicatein (K1890001)-T7 tag into BL21 competent cells.

Protocol:
Transformation was performed using a heat stock method (20mins on ice, 1min in a thermoblock at 42°C, 2mins on ice) into the competent BL21 cells. The bacteria were streaked on LB-agar plates and incubated overnight at 37°C. For those that were not immediately used, store in 4°C refrigerator.

Result:
Transformation for [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta and [7]pET-T7-INP-YFP (K523013)-T7 tag was successful while that of [8]pET-T7-RBS-INP-silicatein (K1890001)-T7 tag failed.

Conclusion:
[6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta and [7]pET-T7-INP-YFP (K523013)-T7 tag were all successful in one run, except for [8]pET-T7-RBS-INP-silicatein (K1890001)-T7 tag, which needed to be transformed again for experimental use. Errors and contaminations needed to be avoided.

**4. PCR**
Date: 05/06/2025
Aim:
To amplify [2]pET-J23119-INP-silicatein, [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM.

Protocol:
First, the volume of DNA required for the reaction was calculated. The primers were diluted to 10 µM if necessary. In the reaction tube, the Master Mix, nuclease-free water, the calculated volume of DNA, and the forward and reverse primers were combined. The total reaction volume was 25 µL. The reaction was run on a PCR machine, which took approximately two hours. Meanwhile, a 1% agarose gel was prepared by dissolving 1 g of agarose in 100 mL of 1× TAE buffer, heating the mixture in a microwave, and adding 3 µL of SYBR Safe dye before pouring it into a casting tray with a wide-tooth comb.

Results:
Nanodrop analysis showed that the concentrations of [1]pET-J23119 INP-silicatein + INP-YFP-csgA, and [7]pET-T7-INP-YFP (K523013)-T7 tag. [7]pET-T7-INP-YFP (K523013)-T7 tag was successful due to high concentration and ideal purity while [1]pET-J23119 INP-silicatein + INP-YFP-csgA failed.

![](https://static.igem.wiki/teams/5685/maynotebook/figure5.webp)

Figure 3.1. PCR processing on the machine

Conclusion:
Plasmids [7]pET-T7-INP-YFP (K523013)-T7 tag were successful, verifying that the bacterial clones contained the correct plasmids with our desired inserts. However, [1]pET-J23119 INP-silicatein + INP-YFP-csgA needed further tests due to extremely and abnormally low concentration. Errors and contaminations needed to be avoided.

**5. Run gel**
Date: 05/06/2025

Aim:
To analyze the success and size of the amplification of [2]pET-J23119-INP-silicatein, [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM by running the products on an agarose gel.

Protocol:
After PCR, the samples were prepared by combining 15 µL of DNA with 3 µL of 6× loading dye for a total volume of 18 µL. The gel was loaded by adding the 18 µL DNA ladder into the first well and the prepared samples into the subsequent wells; an empty well was left between each sample to prevent cross-contamination. The gel was submerged in a tank filled with 1× TAE buffer. Electrophoresis was performed at 80–150 V until the dye front had migrated 75–80% of the gel length, which typically took 1–1.5 hours. Finally, the separated DNA fragments were visualized under UV light.

Results:
The test failed because bands are too close together. But there were band formations.

![](https://static.igem.wiki/teams/5685/maynotebook/figure6.webp)

Figure 3.2. After gel running. Band formation can be seen.

Conclusion:
Visualization of PCR results for plasmids [2]pET-J23119-INP-silicatein, [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM failed, indicating problems during processing. Errors and contaminations needed to be avoided, the procedures needed to be checked again in later tests.

**6. Transformation of plasmid**
Date: 06/06/2025

Aim:
Transform [8]pET-T7-RBS-INP-silicatein (K1890001)-T7 tag into BL21 competent cells.

Protocol:
Transformation was performed using a heat stock method (20mins on ice, 1min in a thermoblock at 42°C, 2mins on ice) into the competent BL21 cells. The bacteria were streaked on LB-agar plates and incubated overnight at 37°C. For those that were not immediately used, store in 4°C refrigerator.

Results:
The transformation of [8]pET-T7-RBS-INP-silicatein (K1890001)-T7 tag failed.

Conclusion:
[8]pET-T7-RBS-INP-silicatein (K1890001)-T7 tag needed to be transformed again for experimental use. Errors and contaminations needed to be avoided.

**7. Midi prep**
Date: 06/06/2025

Aim:
To extract and purify the sequences of [1]pET-J23119 INP-silicatein + INP-YFP-csgA in order to test their concentration.

Protocol:
Plasmid DNA was isolated from a 100 mL bacterial culture. The cells were pelleted by centrifugation (15 min, 4500 × \*g\*), resuspended, lysed with Buffer LYS for 4.5 minutes, and then neutralized. The resulting lysate was clarified by passing it through a pre-equilibrated filter column via gravity flow. The column was washed, and the DNA was eluted with 5 mL of Buffer ELU. The DNA was precipitated by adding isopropanol and centrifuging (20 min, 4500 × g). The pellet was washed with 2 mL of 70% ethanol, dried, and finally resuspended in 80 µL of water for quantification using a Nanodrop.

Results:
The test failed due to extremely and abnormally low concentration of [1]]pET-J23119 INP-silicatein + INP-YFP-csgA DNA.

Conclusion:
[1]pET-J23119 INP-silicatein + INP-YFP-csgA was unsuccessful, indicating that the bacterial clones failed to contain the correct plasmids with our desired inserts. Errors and contaminations needed to be avoided in the future tests.

#### Week 4

---

**1. Gel electrophoresis**

Date: 12/06/2025

Aim:
Transform [1]pET-J23119 INP-silicatein + INP-YFP-csgA into BL21 competent cells.

Protocol:
A 30 mL 1% agarose gel was prepared with 3 µL of SYBR Safe dye and used with approximately 500 mL of 1× TAE buffer. The DNA ladder (Invitrogen 1 kb Plus) was prepared by mixing 1 µL of ladder with 1 µL of loading dye and 8 µL of water, while the samples were prepared by combining 2 µL of DNA with 10 µL of loading dye. These were loaded into the wells, with blank wells left between samples to prevent cross-contamination. Electrophoresis was performed at 120 V on ice for approximately 30 minutes, until the dye front had migrated two-thirds of the way down the gel.

Results:
Gel electrophoresis failed

![](https://static.igem.wiki/teams/5685/wikilok/20250928221414.webp)

Figure 4.1. Visualization of gel electrophoresis results.

![](https://static.igem.wiki/teams/5685/wikilok/20251005223847.webp)

Figure 4.2. Running gel on ince

Conclusion:
High background, smeared bands, and a nearly empty [1]pET-J23119 INP-silicatein + INP-YFP-csgA result suggested overloading PCR products. Another test was required with errors and contamination avoided

**2. Culture bacteria for glycerol stock**
Date: 12/06/2025

Aim:
Pick colony of [2]-[8] and shake in 1 ml LB+Amp overnight

**3. Midi prep**
Date: 13/06/2025

Aim:
To extract and purify the sequences of [1]pET-J23119 INP-silicatein + INP-YFP-csgA, and [7]pET-T7-INP-YFP (K523013)-T7 tag in order to test their concentration.

Protocol:
TPlasmid DNA was isolated from a 100 mL bacterial culture. The cells were pelleted by centrifugation (15 min, 4500 × \*g\*), resuspended, lysed with Buffer LYS for 4.5 minutes, and then neutralized. The resulting lysate was clarified by passing it through a pre-equilibrated filter column via gravity flow. The column was washed, and the DNA was eluted with 5 mL of Buffer ELU. The DNA was precipitated by adding isopropanol and centrifuging (20 min, 4500 × \*g\*). The pellet was washed with 2 mL of 70% ethanol, dried, and finally resuspended in 80 µL of water for quantification using a Nanodrop.

Results:
Nanodrop analysis showed concentrations of [1]pET-J23119 INP-silicatein + INP-YFP-csgA, and [7]pET-T7-INP-YFP (K523013)-T7 tag. Both [1]pET-J23119 INP-silicatein + INP-YFP-csgA, and [7]pET-T7-INP-YFP (K523013)-T7 tag were successful due to high concentration and ideal purity

Table 4.1. The concentrations and purity of the obtained plasmids

![](https://static.igem.wiki/teams/5685/wikilok/20251005224315.webp)

![](https://static.igem.wiki/teams/5685/wikilok/20251005224230.webp)

Figure 4.3. Running Midi results

Conclusion:
Plasmids [1]pET-J23119 INP-silicatein + INP-YFP-csgA, and [7]pET-T7-INP-YFP (K523013)-T7 tag were successful, verifying that the bacterial clones contained the correct plasmids with our desired inserts.

**4. PCR**
Date: 13/06/2025

Aim:
To amplify [2]pET-J23119-INP-silicatein, [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM.

Protocol:
First, the volume of DNA required for the reaction was calculated. The primers were diluted to 10 µM if necessary. In the reaction tube, the Master Mix, nuclease-free water, the calculated volume of DNA, and the forward and reverse primers were combined. The total reaction volume was adjusted to 25 µL. The reaction was run on a PCR machine, which took approximately two hours. Meanwhile, a 1% agarose gel was prepared by dissolving 1 g of agarose in 100 mL of 1× TAE buffer, heating the solution in a microwave, and adding 3 µL of SYBR Safe dye before pouring it into a casting tray with a wide-tooth comb.

Results:
The test failed because nothing was replicated.

![](https://static.igem.wiki/teams/5685/wikilok/20251005224629.webp)

Figure 4.4. Running PCR results

Conclusion:
Either the amplification of DNA was unsuccessful or the visualization of PCR results failed. Errors and contaminations needed to be avoided and the procedures needed to be checked and adjusted in the next tests.

#### Week 5

---

**1. Transformation of plasmid**
Date: 16/06/2025

Aim:
Transform [1]pET-J23119 INP-silicatein + INP-YFP-csgA into BL21 competent cells.

Protocol:
Transformation was performed using a heat-shock method (20 minutes on ice, 1 minute in a thermoblock at 42°C, and 2 minutes on ice) with competent BL21 cells. The bacteria were streaked onto LB-agar plates and incubated overnight at 37°C. Plates not used immediately were stored at 4°C.

Results:
The transformation failed.

Conclusion:

[1]pET-J23119 INP-silicatein + INP-YFP-csgA needed to be transformed again for experimental use. Errors and contaminations needed to be avoided.

**2. Midi prep**
Date: 17/06/2025

Aim:
To extract and purify the sequences of [1]pET-J23119 INP-silicatein + INP-YFP-csgA in order to test its concentration.

Protocol:
A total of 4.5 mL of bacterial culture was pipetted across three tubes. The cells were lysed by resuspending the pellet in 250 µL of Solution 1, followed by the addition of 250 µL of Solution 2 and 350 µL of Solution 3, with gentle inversion after each addition. After centrifugation, the supernatant was applied to a column filter, which was washed with 750 µL of Solution 4. The plasmid DNA was finally eluted with 30 µL of Solution 5 into a new tube via centrifugation.

Results:
The test was successful with ideal concentration acquired.

![](https://static.igem.wiki/teams/5685/wikilok/20251005224903.webp)

Figure 5.1. Running Midi results

Conclusion:
[1]pET-J23119 INP-silicatein + INP-YFP-csgA was successful, verifying that the bacterial clones contained the correct plasmids with our desired insert

**3. qPCR**
Date: 17/06/2025

Aim:
To amplify and measure the amount of our DNA sequences [1]pET-J23119 INP-silicatein + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta, [7]pET-T7-INP-YFP (K523013)-T7 tag, [8]pET-T7-RBS-INP-silicatein (K1890001)-T7 tag in real-time.

Protocol:
A 96-well plate was used. For each well, a 20 µL master mix was prepared by combining 10 µL of SYBR Green dye, 0.2 µL each of the forward and reverse primers, 1 µL (1 ng) of plasmid DNA template (or a negative control consisting of 1 µL of ddH₂O or empty vector), and 8.6 µL of ddH₂O.

Results:

![](https://static.igem.wiki/teams/5685/junenotebook/qpcr2.webp)
![](https://static.igem.wiki/teams/5685/junenotebook/qpcr1.webp)

Figure 5.1. Putting the 96-well plate in the machine         Figure 5.2. Amplification lot when qPCR is completed

![](https://static.igem.wiki/teams/5685/wikilok/the-real-time-pcr-result-of-1-inp-slilicatein-inp-yfp-csga-comparing-with-water.webp)

Figure 5.3. qPCR result of [1]pET-J23119 INP-silicatein + INP-YFP-csgA

![](https://static.igem.wiki/teams/5685/wikilok/the-real-time-pcr-result-of-2-inp-silicatein-comparing-with-water.webp)

Figure 5.4. qPCR result of [2]pET-J23119-INP-silicatein

![](https://static.igem.wiki/teams/5685/wikilok/the-real-time-pcr-result-of-3-inp-yfp-csga-comparing-with-water.webp)

Figure 5.5. qPCR result of [3]pET-T7-INP-YFP(K523013)-csgA

![](https://static.igem.wiki/teams/5685/wikilok/the-real-time-pcr-result-of-4-yded.webp)

Figure 5.6. qPCR result of [4]pET-J23119-ydeD (K4171005)

![](https://static.igem.wiki/teams/5685/silicaengineer/the-real-time-pcr-result-of-5-yham.webp)

Figure 5.7. qPCR result of [5]pET-J23119-yhaM

![](https://static.igem.wiki/teams/5685/silicaengineer/the-real-time-pcr-result-of-6-t7-sulfite-reductase-comparing-with-water.webp)

Figure 5.8. qPCR result of [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta

![](https://static.igem.wiki/teams/5685/silicaengineer/the-real-time-pcr-result-of-7-t7-inp-yfp.webp)

Figure 5.9. qPCR result of [7]pET-T7-INP-YFP (K523013)-T7 tag

![](https://static.igem.wiki/teams/5685/silicaengineer/the-real-time-pcr-result-of-8-t7-silicatein-comparing-with-water.webp)

Figure 5.10. qPCR result of [8]pET-T7-RBS-INP-silicatein (K1890001)-T7 tag

Conclusion:
The qPCR results for [1]pET-J23119 INP-silicatein + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta, [7]pET-T7-INP-YFP (K523013)-T7 tag, [8]pET-T7-RBS-INP-silicatein (K1890001)-T7 tag indicated that our plasmids were replicated successfully with ideal and significant fold changes.

**4. Protein extraction**

Date: 18-19/06/2025

Aim:
 To extract and prepare soluble proteins from [1]pET-J23119 INP-silicatein + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM for subsequent analysis.

Protocol:
Cells were pelleted (4000 rpm, 15 min), lysed in 1 mL ice-cold RIPA buffer (30 min on ice), and centrifuged (16,000 x g, 20 min, 4°C). The supernatant was collected and stored at -20°C. Additionally, 500 mL of LB br /oth and 1000 mL of MOPS-SDS running buffer were prepared.

#### Week 6

---

**1. Western blot**
Date: 25-26/06/2025

Aim:
To validate the expression and confirm the size of [1]pET-J23119 INP-silicatein + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM.

Protocol:
Samples were denatured by boiling at 95°C for 10 min in SDS buffer. Proteins were separated by electrophoresis at 100 V for 1.5 hours (adjusted to 80 V if needed) with the tank placed on ice, and then transferred to a membrane at 200 mA for 2 hours at 4°C. The membrane was blocked for 1.5 hours, incubated with the secondary antibody for 1 hour, and washed three times for 10 min each with TBST. Specific proteins were detected by scanning the membrane.

Results:
Images of plasmid [1]pET-J23119 INP-silicatein + INP-YFP-csgA, and [3]pET-T7-INP-YFP(K523013)-csgA could be successfully observed while that of others could not.

![](https://static.igem.wiki/teams/5685/wikilok/20251005230033.webp)

Figure 6.1. Western blot visualization on screen

![](https://static.igem.wiki/teams/5685/wikilok/20251005230009.webp)

Figure 6.2. Western blot visualization

Conclusion:
[1]pET-J23119 INP-silicatein + INP-YFP-csgA, and [3]pET-T7-INP-YFP(K523013)-csgA that used GFP succeeded in band visualization while others using flag tag failed. The failed groups might use antibodies too many times.

**2. Preparation for flushing test for bacteria adhesion**
Date: 25/06/2025

Aim:
Prepare 100mL of HNO₃ with pH 3, 100mL of HNO₃ with pH 5, and 100mL of NaOH with pH 9.

#### Week 7

---

**1. Western Blot**
Date: 30/06/2025
Aim:
To validate the expression and confirm the size of [1]pET-J23119 INP-silicatein + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM.

Protocol:
Samples were denatured by boiling at 95°C for 10 minutes in SDS buffer. Proteins were separated via SDS-PAGE using SDS running buffer at 160 V for 45 minutes (adjusted to 80 V if distortion occurred) with the tank placed on ice. The gel was then assembled into a transfer stack with a methanol-activated membrane and transferred at 200 mA for 2 hours using transfer buffer. The entire transfer process was conducted with an ice block to maintain a low temperature.

**2. Coomassie blue**
Date: 30/06/2025
Aim:
To visually detect and assess the total protein profile of [1]pET-J23119 INP-silicatein + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM.

Protocol:
The gel was washed in water for 5 minutes per wash, repeated 3 times. It was then stained with 50 mL of staining solution per gel for 1 hour with gentle shaking. Finally, the gel was rinsed with water for 30 minutes and could be stored in water.

![](https://static.igem.wiki/teams/5685/julynotebook/com-blue.webp)

Figure 7.1. Coomassie blue staining.

Results:
The western blot failed.

Conclusion:
No protein bands were visible—only the tracking dye—indicating no protein loaded, poor transfer or staining. Errors and contamination needed to be avoided in future tests.

 **3.Preparation for IPTG Stock Solution**
Date: 01/07/2025
Aim:
Bacteria carrying T7 tag require IPTG solution to remove a repressor from the lac operon to induce gene expression.

Protocol:
Prepare 4 mL of distilled water in a suitable container. Add 1.191g of IPTG (isopropyl-β-D-thiogalactopyranoside) to the solution. Add distilled water until the volume is 5mL. Sterilize by passing it through a 0.22µm disposable filter. Dispense the solution into 1ml aliquots and store them at -20°C.
 **4.Flushing test for bacteria adhesion**
Date: 01/07/2025
Aim:
To verify the E.coli which carry the csgA gene resist rinsing with solutions, creating adhesion between the building surface.

Protocol:
1st, 2nd and 3rd time E.coli flushing test for adhesion using H2SO4, PBS and NaOH solutions of different pH values to flush the slide adhere with bacterial solution. Photograph with fluorescence microscope for subsequent colony counts with imageJ.

![](https://static.igem.wiki/teams/5685/julynotebook/262f7e6b8c496e0697ff99eec5734429-1.webp)

Figure 7.2. Photograph colonies with fluorescence microscope.

Results:

![](https://static.igem.wiki/teams/5685/julynotebook/flushing-test-result-p1.webp)

Figure 7.3. DIC and YFP images of BL21-INP-silicatein-INP-YFP-csgA without flushing (100x).

![](https://static.igem.wiki/teams/5685/julynotebook/flushing-test-result-p2.webp)

Figure 7.4. DIC and YFP images of BL21-INP-silicatein-INP-YFP-csgA flushed with pH3 H2SO4 (100x).

Conclusion:
A significant number of bacteria retain on the slide surface even after flushing with solution across a range of pH conditions (Figure 7.3). This result is consistent across experimental bacteria: BL21-INP-YFP-csgA, pET-T7-INP-YFP-T7 tag. Also, low pH enhances adhesion of protein binding domains (CsgA).
 **5.Preparation of cysteine destruction test for prevention**
Date: 02/07/2025 & 03/07/2025
Aim:
Prepare Reagent 2 of Cysteine content assay kit and shake BL21 stock, BL21-pET-J23119-ydeD (K4171005) and BL21-pET-J23119-yhaM.

Protocol:
We chose to use BL21 bacteria as our control group. There are a total of 6 experimental groups, including 3 groups with preadded cysteine and the other 3 without. With final concentrations of 2.862mM Ampicillin and 0.5mM of cysteine in LB broth, the Cysteine-LB-Amp broth is prepared. With final concentrations of 2.862mM Ampicillin in LB broth, the LB-Amp broth is prepared. Add 30μL (1:100 v:v) of bacteria stock (BL21, BL21-pET-J23119-ydeD (K4171005) and BL21-pET-J23119-yhaM) to 3mL of two broths each. Then incubate them at 37°C 220rpm overnight.
 **6. Preparation for Test for the effect of Sulfite and Sulfate on bacterial growth**
Date: 02/07/2025
Aim:
Preparing for 10mM sulfite stock solution, 10mM sulfate stock solution, 5mM sulfite broth, 5mM sulfate broth, control broth.

Protocol:
Sulfite stock solution prep: 0.5g sodium Sulfite powder, 396.73ml ddH2O. Sulfate stock solution prep: 0.833ml of 3M sulfuric acid into 250ml volumetric flask, make up the 250ml mark with ddH2O. Sulfite broth: 10mL LB into 15ml centrifuge tube, then add 3ml Sulfite stock solution and 10ul ampicillin. Sulfate broth: 10mL LB into 15ml centrifuge tube, then add 3ml sulfate stock solution and 10ul ampicillin. Control broth: 13 ml LB + 10ul ampicillin as control. Culture 6 in the 3 broths and incubate overnight at 37°C 150 rpm.
 **7. Transformation of plasmid**
Date: 02/07/2025
Aim:
Transform pET11a into BL21 competent cells.

Protocol:
Transformation was performed using a heat stock method ( 20mins on ice, 1min in a thermoblock at 42°C, 2mins on ice) into the competent BL21 cells. The bacteria were streaked on LB-agar plates and incubated overnight at 37°C. For those which are not immediately used, store in 4°C refrigerator.

Results:
Colonies grow successfully after transformation of pET11a into BL21 competent cells.

![](https://static.igem.wiki/teams/5685/julynotebook/8e37f6baac8abbf89a5e9c23de09b66b.webp)

Figure 7.5. Growth of bacteria BL21-pET11a after transformation.

 **8. Preparation of stocking plasmid**
Date: 02/07/2025
Aim:
Culturing pET-J23119-INP-silicatein colonies from agar plate in Amp-LB broth to prepare for stocking the bacteria.

Protocol:
Label the 14 mL Polystyrene Round-Bottom Tube with “[2] BL21”. Pick and pipette 1 colony on [2] BL21 plate with a pipette. Place the “[2] BL21” tube into the incubator at 220 rpm and 37°C overnight.
 **9. Cysteine destruction test for prevention**
Date: 03/07/2025 & 04/07/2025
Aim:
Test whether BL21-pET-J23119-ydeD (K4171005) and BL21-pET-J23119-yhaM reduce cysteine amount.

Protocol:
The test was carried out with Sangon cysteine content assay kit. By testing standard cysteine solution with concentrations of 2, 1, 0.5, 0.25, 0.125, 0.0625 mM, we obtained the standardised best-fit of different concentrations of cysteine by testing its absorbance at 600nm with the reagents in the Sangon cysteine content assay kit. Pipette 0.2mL of the incubated bacteria and 0.3mL of the Cysteine extract liquid. Centrifuge at 11000 rpm at 4°C for 10 minutes. Remove the supernatant. Preheat the spectrophotometer (600 nm) for 30 minutes.
Test the groups below with the Sangon cysteine content assay kit and record the absorbance:

* [4] YdeD BL21 + Cys + Amp test
* [4] YdeD BL21 + Amp test
* [5] yhaM BL21 + Cys + Amp test
* [5] yhaM BL21 + Amp test
* BL21 stock + Cys + Amp test
* BL21 stock + Amp test
* ddH2O blank test
* 2 mM Cys Standard
* 1 mM Cys Standard
* 0.5 mM Cys Standard
* 0.25 mM Cys Standard
* 0.125 mM Cys Standard
* 0.0625 mM Cys Standard

Expected results are the lower absorbance at 600nm for the experimental groups.

Results:
The cysteine destruction test failed.

![](https://static.igem.wiki/teams/5685/julynotebook/cys.webp)

Figure 7.6. Standardised best-fit of different absorbance at 600nm against corresponding concentrations of cysteine.

Table 7.1. Absorbance at 600nm for the groups on 03/07/2025.

![](https://static.igem.wiki/teams/5685/julynotebook/table-7-1.webp)

Table 7.2. Absorbance at 600nm for the 6 experimental groups on 04/07/2025.

![](https://static.igem.wiki/teams/5685/julynotebook/table-7-2.webp)

Conclusion:
The tests on two days are both failed due to the limited amount of cysteine, causing the concentrations of cysteine in “BL21 stock + Cys + Amp test” even lower than the experimental groups. We increased the amount of cysteine added in the following tests.
 **10.Preparation of cysteine destruction test for prevention**
Date: 06/07/2025
Aim: Shake BL21 stock, BL21-pET-J23119-ydeD (K4171005) and BL21-pET-J23119-yhaM.

Protocol:
We chose to use BL21 bacteria as our control group. There are a total of 9 experimental groups, including 3 groups with preadded 1mM cysteine, 3 groups with preadded 2mM cysteine and the other 3 without. With final concentrations of 2.862mM Ampicillin and 1mM of cysteine in LB broth, the 1mM-Cysteine-LB-Amp broth is prepared. With final concentrations of 2.862mM Ampicillin and 2mM of cysteine in LB broth, the 1mM-Cysteine-LB-Amp broth is prepared. With final concentrations of 2.862mM Ampicillin in LB broth, the LB-Amp broth is prepared. Add 30μL (1:100 v:v) of bacteria stock (BL21, BL21-pET-J23119-ydeD (K4171005) and BL21-pET-J23119-yhaM) to 3mL of three broths each. Then incubate them at 37°C 220rpm overnight.

#### Week 8

---

**1. Preparing LB agar and LB broth**
Date: 07/07/2025

Protocol:
Measure 10g of LB agar powder and 500mL of MiniQ water. Add 10g LB agar powder to 500mL MiniQ and mix thoroughly. Repeat the process to make four jars of LB broth.
 **2. Preparation for testing Sulfite and sulfate on ydeD, yhaM and sulfite reductase bacterial growth**
Date: 07/07/2025
Aim:
Shake [4] pET-ydeD (K4171005), [5] pET-yhaM, [6] pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta for bacterial growth test.

![](https://static.igem.wiki/teams/5685/julynotebook/figure-8-0-0.webp)

Figure 8.1. Shaking [4][5][6] for bacterial growth test.

![](https://static.igem.wiki/teams/5685/julynotebook/figure-8-0.webp)

Figure 8.2. Shaking [4][5][6] for bacterial growth test.

 **3. Cysteine destruction test for BL21 pET-J23119-ydeD (K4171005) and BL21-pET-J23119-yhaM**
Date: 08/07/2025 & 09/07/2025
Aim:
Test whether BL21-pET-J23119-ydeD (K4171005) and BL21-pET-J23119-yhaM reduce cysteine amount.

Protocol:
The test was carried out with Sangon cysteine content assay kit. By testing standard cysteine solution with concentrations of 2, 1, 0.5, 0.25, 0.125, 0.0625 mM, we obtained the standardised best-fit of different concentrations of cysteine by testing its absorbance at 600nm with the reagents in the Sangon cysteine content assay kit. Pipette 0.2mL of the incubated bacteria and 0.3mL of the Cysteine extract liquid. Centrifuge at 11000 rpm at 4°C for 10 minutes. Remove the supernatant. Preheat the spectrophotometer (600 nm) for 30 minutes.

Table 8.1. Operation table for cysteine assay kit.

![](https://static.igem.wiki/teams/5685/julynotebook/table-8-1.webp)

![](https://static.igem.wiki/teams/5685/julynotebook/figure-8-1.webp)

Figure 8.3. Test tubes with experimental solutions.

![](https://static.igem.wiki/teams/5685/julynotebook/figure-8-2.webp)

Figure 8.4. Test tubes with experimental solutions.

Results: ydeD[4] and yhaM[5] can successfully reduce cysteine amounts.

Table 8.2. Concentrations of cysteine and absorbance at 600nm for the 9 experimental groups on 08/07/2025.

![](https://static.igem.wiki/teams/5685/julynotebook/table-8-2.webp)

Table 8.3. Concentrations of cysteine and absorbance at 600nm for the 9 experimental groups on 08/07/2025.

![](https://static.igem.wiki/teams/5685/julynotebook/table-8-3.webp)

Conclusion:
Lower absorbance (600nm) and concentration in BL21-pET-J23119-ydeD (K4171005) and BL21-pET-J23119-yhaM strains compared to control (BL21 pET11a) which verify ydeD[4] and yhaM[5] can successfully reduce cysteine amounts. We repeated the experiments several times afterwards to calculate the percentage of errors.
 **4. Preparation of cysteine destruction test of BL21-pET-J23119-ydeD (K4171005) and BL21-pET-J23119-yhaM**
Date:08/07/2025
Aim:
Shaking BL21, BL21-pET-J23119-ydeD (K4171005) and BL21-pET-J23119-yhaM stock for cysteine destruction test.

Protocol:
We chose to use BL21 bacteria as our control group. There are a total of 9 experimental groups, including 3 groups with preadded 1mM cysteine, 3 groups with preadded 2mM cysteine and the other 3 without. With final concentrations of 2.862mM Ampicillin and 1mM of cysteine in LB broth, the 1mM-Cysteine-LB-Amp broth is prepared. With final concentrations of 2.862mM Ampicillin and 2mM of cysteine in LB broth, the 1mM-Cysteine-LB-Amp broth is prepared. With final concentrations of 2.862mM Ampicillin in LB broth, the LB-Amp broth is prepared. Add 30μL (1:100 v:v) of bacteria stock (BL21, BL21-pET-J23119-ydeD (K4171005) and BL21-pET-J23119-yhaM) to 3mL of three broths each. Then incubate them at 37°C 220rpm overnight.
 **5. Testing sulfuric acid on ydeD, yhaM and sulfite reductase bacterial growth**
Date: 08/07/2025
Aim: Test to investigate the effect of sulfite and sulfate on of bacteria growth.

* [4] pET-ydeD (K4171005)
* [5] pET-yhaM
* [6] pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta

[4] pET-ydeD (K4171005)
[5] pET-yhaM
[6] pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta

Protocol:
Take tubes prepared on 7/7 out of the incubator. Pipette 30uL LB with cultures out from tubes, add them into the LB broth with sulfuric acid. Measure OD600 every hour for 5 hours. Until the OD600 of the LB broth of [6] reaches 0.6, add IPTG.

![](https://static.igem.wiki/teams/5685/julynotebook/figure-8-5.webp)

Figure 8.5. Measuring absorbance at 600nm.

Results:
The bacterial growth test failed.

Table 8.4. Absorbance at 600nm(A) over 5 hours.

![](https://static.igem.wiki/teams/5685/julynotebook/table-8-4.webp)

![](https://static.igem.wiki/teams/5685/julynotebook/figure-8-6.webp)

Figure 8.6. Curves (OD vs. time) for ydeD[4], yhaM[5], sulfite reductase[6] growth with 5mM concentrations of sulfate added.

Conclusion:
The OD600 value in experimental broth with 5mM sulfuric acid did not show any obvious increase, indicating that the concentration 5mM sulfuric acid is too high, and it affects the growth of bacteria BL21-pET11a, ydeD[4], yhaM[5], sulfite reductase[6]. Therefore, we decided to test for a lower concentration of sulfuric acid, sodium sulfite and sodium sulfate in the following weeks.
 **6. Testing the effect of sulfate and sulfite on BL21-pET11a growth**
Date: 09/07/2025
Aim: Testing the effect of different concentration of sulfate and sulfite on BL21-pET11a growth.

Protocol:
Prepare LB broth with sulfate, LB broth with sulfite in different concentration and LB broth(control). Take tubes prepared on 8/7 out of the incubator. Pipette 30uL LB with cultures out from tubes, add them into the LB broth(control), LB broth with different concentrations of sulfate and sulfite respectively. Measure OD600 every hour for 5 hours.

![](https://static.igem.wiki/teams/5685/julynotebook/figure-8-7.webp)

Figure 8.7. Measuring absorbance at 600nm.

Results:
Successful growth of bacteria BL21-pET11a under different concentrations of sulfate.

Table 8.5. Absorbance at 600nm(A) of BL21-pET11a with different concentrations of sulfate over 5 hours.

![](https://static.igem.wiki/teams/5685/julynotebook/table-8-5.webp)

![](https://static.igem.wiki/teams/5685/julynotebook/figure-8-8.webp)

Figure 8.8. Curves (OD vs. time) for BL21-pET11a growth with different concentrations of sulfate added.

Table 8.6. Absorbance at 600nm(A) of BL21-pET11a with different concentrations of sulfite over 5 hours.

![](https://static.igem.wiki/teams/5685/julynotebook/table-8-6.webp)

![](https://static.igem.wiki/teams/5685/julynotebook/figure-8-9.webp)

Figure 8.9. Curves (OD vs. time) for BL21-pET11a growth with different concentrations of sulfite added.

Conclusion:
Absorbance at 600nm(A) of BL21-pET11a with different concentrations of sulfate (0.01mM, 0.1mM and 1mM) increases significantly over 5 hours, indicating that the concentration does not affect the growth of bacteria BL21-pET11a.

#### Week 9

---

**1. Transformation of [4]pET-J23119-ydeD (K4171005)**
Date: 14/07/2025
Aim:
Transform [4]BL21-pET-J23119-ydeD (K4171005) into BL21 competent cells.

Protocol:
Transformation was performed using a heat stock method ( 20mins on ice, 1min in a thermoblock at 42°C, 2mins on ice) into the competent BL21 cells. The bacteria were streaked on LB-agar plates and incubated overnight at 37°C. For those which are not immediately used, store in a 4°C refrigerator.
Results: [4]BL21-pET-J23119-ydeD (K4171005) was successfully transformed into BL21 competent cells.
**2. Preparation of cysteine destruction test for prevention**
Date: 14/07/2025
Aim:
Shaking BL21 stock, [4]BL21-pET-J23119-ydeD (K4171005) and [5]BL21-pET-J23119-yhaM.

Protocol:
We chose to use BL21 bacteria as our control group. There are a total of 9 experimental groups, including 3 groups with preadded 1mM cysteine, 3 groups with preadded 2mM cysteine and the other 3 without. With final concentrations of 2.862mM Ampicillin and 1mM of cysteine in LB broth, the 1mM-Cysteine-LB-Amp broth is prepared. With final concentrations of 2.862mM Ampicillin and 2mM of cysteine in LB broth, the 1mM-Cysteine-LB-Amp broth is prepared. With final concentrations of 2.862mM Ampicillin in LB broth, the LB-Amp broth is prepared. Add 30μL (1:100 v:v) of bacteria stock (BL21, BL21-pET-J23119-ydeD (K4171005) and BL21-pET-J23119-yhaM) to 3mL of three broths each. Then incubate them at 37°C 220rpm overnight.
**3. Cysteine destruction test for prevention**
Date: 15/07/2025
Aim:
Testing whether [4]pET-J23119-ydeD (K4171005) and [5]pET-J23119-yhaM are capable of lowering the amount of cysteine.

Protocol:
Using Cysteine Content essay kit
Pipette 0.2mL of the samples and 0.3mL of the kit liquid. Centrifuge at 11000 rpm at 4°C for 10 minutes. Remove the supernatant. Preheat the spectrophotometer (600 nm) for 30 minutes. Add the solutions respectively according to the operation table (Table 1.), and test the absorbance at 600nm after waiting for 15 minutes.

Table 9.1. Operation table for cysteine assay kit

![](https://static.igem.wiki/teams/5685/julynotebook/table-11.webp)

![](https://static.igem.wiki/teams/5685/julynotebook/fig-30.webp)

Figure 9.1. Testing OD600 values for samples after adding in kit solutions.

Results:

Table 9.2. Concentrations of cysteine and absorbance at 600nm for the 9 experimental groups on 15/07/2025

![](https://static.igem.wiki/teams/5685/julynotebook/table-12.webp)

![](https://static.igem.wiki/teams/5685/julynotebook/fig-31.webp)

Figure 9.2. Cysteine amounts of different samples after adding cysteine before incubating

Conclusion:
Lower absorbance (600nm) and concentration in [4]pET-J23119-ydeD (K4171005) and [5]pET-J23119-yhaM strains compared to control (BL21 pET11a) which verify they can successfully reduce cysteine amounts. We repeated the experiments several times to calculate the error bar shown in Figure 31.

#### Week 10

---

**1. Preparation of flushing test**
Date: 21/07/2025
Aim:
Shaking [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [3]pET-T7-INP-YFP(K523013)-csgA, [7]pET-T7-INP-YFP (K523013)-T7 tag, and BL21-pET11a stock for flushing test
**2. Flushing test for bacteria adhesion**
Date: 22/07/2025
Aim:
To verify the E.coli which carry the csgA gene resist rinsing with solutions, creating adhesion between the building surface.

Protocol:
1st, 2nd and 3rd time E.coli flushing test for adhesion using H2SO4, PBS and NaOH solutions of different pH values to flush the slide adhere with bacterial solution. Photograph with fluorescence microscope for subsequent colony counts with imageJ.

![](https://static.igem.wiki/teams/5685/julynotebook/fig-32.webp)

Figure 10.1. Bacterial culture on slides.

Results:

![](https://static.igem.wiki/teams/5685/julynotebook/fig-33.webp)

Figure 10.2. DIC and YFP images of BL21-T7-INP-YFP without flushing (100x)

![](https://static.igem.wiki/teams/5685/julynotebook/fig-34.webp)

Figure 10.3. DIC and YFP images of BL21-T7-INP-YFP

Conclusion:
After rinsing at different pH conditions, a significant portion of bacterial cells remained on the slide surface. Ice nucleation protein (INP) enables the colonies to adhere firmly to the surface, preventing them from falling off. This strong adhesion was observed in all samples.
**3. IPTG Induction + Protein Extraction [4]pET-J23119-ydeD (K4171005) [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta**
Date: 22/07/2025
Aim:
IPTG is added to bacterial samples to regulate protein synthesis of sulfite reductase, proteins of [4]pET-J23119-ydeD (K4171005) and [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta are extracted for Western Blot.

Protocol:
IPTG is added according to a ratio of 1:1000 (final concentration: 0.05mM). Bacterial samples are centrifuged and cell pellets are washed with 1mL 4C PBS after that. Samples are then sonified and collected.
![](https://static.igem.wiki/teams/5685/julynotebook/fig-35.webp)

Figure 10.4. Sonicating the samples

**4. Western Blot**
Date: 24/07/2025 and 25/07/2025
Aim:
Western Blot is performed to verify the expression of proteins in [4]pET-J23119-ydeD (K4171005) and [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta.
Protocol:
Samples are boiled at 95C for 10 min, then gel and core is inserted to the transfer tank. Comb is removed and a running buffer is added. Wells are flushed, and 10uL samples are loaded to gel perpendicularly, adding a running buffer subsequently. The entire tank is put in ice and cover on and run: 160V for 45 min. When done, the buffer is poured away and unwanted parts of the gel are cutted out. The membrane is submerged in methanol, wait for 5 minutes then 1x transfer buffer. Add some solution to the transfer sandwich. Submerge gel in solution. Assemble transfer sandwich by placing sponge then filter paper then gel then membrane then filter paper then sponge. Close the sandwich. Insert sandwich to the gel holder cassette. Insert transfer cassette to the tank and add transfer buffer to ‘blotting’ line. Place an ice block in the tank. Place the transfer tank in a 4°C cold room and run overnight at 40mA. Remove membrane and cut the membrane between two makers and soak it in a blocking buffer. Place on an orbital shaker and shake for 1.5 hours. Wash 3 times with 1X TBST and place on an orbital shaker and shake for 10 min each time. Secondary antibody in anti mouse and anti rabbit respectively for 1 hour at room temperature. Wash 3 times 1X TBST for 10 min each time. Clean the imaging scanning bed with 70% ethanol using a cotton lint-free cloth. Scan membranes in the imaging system. Remove the membranes from the scan bed and clean the imaging scan bed with 70% ethanol using a cotton lint-free cloth. Detection is performed.
Results:
Western blot failed.
Conclusion:
Western blot due to the method of extraction. We decided to use other method for protein extraction.
**5. Running buffer for Western Blot preparation**
Date: 24/07/2025
Aim:
Prepare running buffer for Western Blot.

Protocol:
Powder and 1L ddH2O are added together and swirl until completely dissolved.
Results:
Running buffer was prepared successfully.
**6. 1x Transfer buffer for Western Blot preparation**
Date: 24/07/2025
Aim:
Prepare 1x Transfer buffer for Western Blot.

Protocol:
100mL 10x transfer buffer, 700mL ddH2O and 200mL methanol are mixed together.
Results:
1x Transfer buffer was prepared successfully.

#### Week 11

---

**1. Colony qPCR**
Date: 28/07/2025
Protocol:
Add 1mL LB, 1uL Amp, 10uL bacterial stock solution. Incubate at 37C 220 rpm overnight. Transfer 100uL incubated samples to ep tubes. Centrifuge and discard supernatant. Add 100uL ddH2O and resuspend. Boil at 95C for 10 mins and centrifuge. Add: 8ul H2O then 2uL centrifuged samples then 1.25 ul fwd primer then 1.25 ul rev primer then 12.5uL master mix to PCR tubes. Select the tm50 program and run the machine. Transfer 5 ul collected samples to PCR tubes as template. Add 1uL loading dye to the tubes. Pipette tube content perpendicularly into the wells of the gel. Add 4uL ladder to the remaining well, add 1x TAE until gel submerged. Run at 80V.
Results:
Bands visible for all samples, ergo no problem with transformation.
**2. Preparation for the effect of different concentration of sulfate and sulfite on BL21-pET11a growth**
Date: 28/07/2025
Aim: Shaking BL21-pET11a stock.
**3. Transformation of plasmid**
Date: 29/07/2025
Aim:
Transform pET-T7-INP-YFP-csgA into BL21 competent cells.
Protocols:
Transformation was performed using a heat stock method ( 20mins on ice, 1min in a thermoblock at 42°C, 2mins on ice) into the competent BL21 cells. The bacteria were streaked on LB-agar plates and incubated overnight at 37°C. For those which are not immediately used, store in 4°C refrigerator.
Results:
pET-T7-INP-YFP-csgA was successfully transformed into BL21 competent cells.
**4. Testing the effect of sulfate and sulfite on BL21-pET11a growth**
Date: 29/07/2025
Aim:
Testing the effect of different concentration of sulfate and sulfite on BL21-pET11a growth.
Protocol:
Prepare LB broth with sulfate, LB broth with sulfite in different concentration and LB broth(control). Take tubes prepared on 8/7 out of the incubator. Pipette 30uL LB with cultures out from tubes, add them into the LB broth(control), LB broth with different concentrations of sulfate and sulfite respectively. Measure OD600 every hour for 5 hours.
![](https://static.igem.wiki/teams/5685/julynotebook/fig-11-1.webp)

Figure 11.1 Adding sulfite and sulfate into LB broth respectively

![](https://static.igem.wiki/teams/5685/julynotebook/fig-11-2.webp)

Figure 11.2 Measuring OD600 values of samples

Results:
Although the OD600 values of BL21-pET11a varied with different sulfite concentrations over the 5-hour period, all conditions exhibited similar rates of increase.

Table 11.1 Sulfite group, OD600 values of BL21-pET11a with different concentrations over 5 hours

![](https://static.igem.wiki/teams/5685/julynotebook/tab-11-1.webp)
![](https://static.igem.wiki/teams/5685/julynotebook/fig-11-3.webp)

Figure 11.3 Sulfite group, curves (OD vs. time) for BL21-pET11a growth with different concentrations of sulfate added

Table 11.2 Sulfate group, OD600 values of BL21-pET11a with different concentrations over 5 hours

![](https://static.igem.wiki/teams/5685/julynotebook/tab-11-2.webp)
![](https://static.igem.wiki/teams/5685/julynotebook/fig-11-4.webp)

Figure 11.4 Sulfate group, curves (OD vs. time) for BL21-pET11a growth with different concentrations of sulfate added

Conclusion:
The similar growth curves of OD600 indicate that different concentrations of sulfite and sulfate (0.01mM, 0.1mM and 1mM) do not affect the growth of bacteria BL21-pET11a.
**5. Flushing test for bacteria adhesion**
Date: 30/07/2025
Aim:
To verify the E.coli which carry the csgA gene resist rinsing with solutions, creating adhesion between the building surface.
Protocol:
3rd time E.coli flushing test for adhesion using H2SO4, PBS and NaOH solutions of different pH values to flush the slide adhere with bacterial solution. Photograph with fluorescence microscope for subsequent colony counts with imageJ.
Results:
The control (pET11a) initially had many cells attached (2127 colonies), but most were easily washed away, especially by neutral and basic solutions. This shows its adhesion was weak. The plasmids of [1] and [3] behaved differently. While fewer cells attached initially, they held on much tighter during rinsing. plasmid [1] showed its strongest adhesion after rinsing at neutral and basic pH (7 and 9). plasmid [3] resisted the acidic rinse (pH 3) best, with more cells remaining after washing.plasmid [7] showed very low adhesion overall.
![](https://static.igem.wiki/teams/5685/julynotebook/fig-11-5.webp)

Figure 11.5 DIC and YFP images of BL21-T7-INP-YFP-csgA flushed with pH3 H2SO4 (100x)

![](https://static.igem.wiki/teams/5685/julynotebook/fig-11-6.webp)

Figure 11.6 DIC and YFP images of BL21-T7-INP-YFP-csgA flushed with pH7.4 PBS (100x)

Conclusion:
A substantial proportion of bacterial cells persist on the slide surface following flushing under various pH conditions. This robust adherence is observed in both tested bacteria (BL21-INP-YFP-csgA and pET-T7-INP-YFP-T7 tag). Acidic conditions significantly strengthen the adhesion mediated by the CsgA protein domains.
**6. Preparation for the effect of different concentration of sulfate and sulfite on BL21-pET11a growth**
Date: 31/07/2025
Aim:
Shaking BL21-pET11a stock.
**7. IPTG Induction + Protein Extraction [3]pET-J23119-INP-YFP-csgA [4]pET-J23119-ydeD (K4171005) [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta**
Date: 31/07/2025
Aim:
IPTG is added to bacterial samples to regulate protein synthesis of sulfite reductase, proteins of [3]pET-J23119-INP-YFP-csgA, [4]pET-J23119-ydeD (K4171005) and [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta are extracted for Western Blot.
Protocol:
IPTG is added according to a ratio of 1:1000 (final concentration: 0.05mM). Bacterial samples are centrifuged and cell pellets are washed with 1mL 4C PBS after that. Samples are then sonified and collected.
**8. Protein Extraction [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005) [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta**
Date: 01/08/2025
Aim:
Proteins of [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005) and [6]pET-T7-RBS-NSP4-sulfite reductase alpha-NSP4-sulfite reductase beta are extracted for Western Blot.
Protocol:
Add start culture to 15 mL LB with 500 ML Amp., shake at 220 rpm 37°C. Check OD600 2 hrs after start. Stop when OD600=0.6-0.8. Use 0.5M IPTG stock solution to induce (1/1000, v/v) bacterial culture for 3 hours. Take 2 mL bacterial culture, 1mL into each ep tube. Centrifuge at 4500 rpm for 5 minutes. Discard supernatant, wash cell pellets with 200uL 4C PBS. Centrifuge at 4500 rpm for 5 minutes, wash cell pellets again with 1mL 4C PBS. Store at 4C. Sonify the cell mixture. Use a 400mL beaker to discard ~400mL water in the tank and make up the volume by putting ice in until the volume in the tank reaches the „water level“ line. Put the ep tubes into the gear container. Ensure the content is submerged in the ice-water mixture. Tighten the container and put it in the tank. Make sure the gear is engaged. Set the machine to 60s run and 60s rest for 5 cycles. Put in ice when collecting sonified samples.
**9. Western blot of [2]pET-J23119-INP-silicatein, [3]pET-T7-INP-YFP(K523013)-csgA, [4]pET-J23119-ydeD (K4171005), and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**
Date: 01/08/2025
Aim:
Detect our specific proteins in the four engineered E. Coli.
Protocol:
Boil samples at 95C for 10 minutes. Insert core to transfer tank. Insert gel to core. Remove the comb and check whether wells are upright. Add SDS buffer to core. Flush wells with pipette gently and perpendicularly to remove impurities. Load 10ul sample to gel perpendicularly and slowly . (3.5 ul marker). Add Running buffer to reference line (2 for 1-2 gels/4 for 3-4 gels). Put the entire tank in ice. Put cover on and run: 160V for 45 min. Check for effervescence in the buffer (verify whether the device is running). When done, pour the buffer away and cut out unwanted parts of the gel (wells, excess part…). Submerge membrane in methanol, wait for ~5 mins then 1x transfer buffer. Add some solution to the transfer sandwich. Submerge gel in solution. Assemble transfer sandwich by placing sponge then filter paper then gel then membrane then filter paper then sponge. Close the sandwich. Insert sandwich to the gel holder cassette. Insert transfer cassette to the tank and add transfer buffer to ‘blotting’ line. Place an ice block in the tank. Place the tank on ice. Run at 200mA for 2 hrs. Remove membrane from assembly sandwich. Wash membrane with 1x TBST. Soak membrane in blocking buffer and place on orbital shaker. Shake for 1.5 hr. Wash 3 times with 1X TBST and place on an orbital shaker. Shake at 70 rpm for 10 min each time. Add secondary antibodies (anti mouse/anti rabbit) respectively. Shake for 1 hour at 70 rpm, room temperature. Wash 3 times 1X TBST. Shake at 70 rpm for 5 min each time. Clean the imaging scanning bed with 70% ethanol using a cotton lint-free cloth. Scan membranes in the imaging system. Remove the membranes from the scan bed and clean the imaging scan bed with 70% ethanol using a cotton lint-free cloth,then do the detection.
Results:

![](https://static.igem.wiki/teams/5685/julynotebook/fig-11-7.webp)

Figure 11.7 Successful western blot of [2] INP-silicatein (BBa\_K1890001)

![](https://static.igem.wiki/teams/5685/julynotebook/fig-11-8.webp)

Figure 11.8 Successful western blot of [3] T7-INP-YFP-csgA

Conclusion:
Our target proteins of [2] INP-silicatein (BBa\_K1890001) and [3] T7-INP-YFP-csgA were successfully detected and observed. Though the target proteins of [4]pET-J23119-ydeD (K4171005), and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta were not shown on the detection, our functional tests (cysteine destruction test and sulfite reduction test) prove their existence.
**10. Preparation of blocking buffer for Western blot**
Date: 01/08/2025
Aim:
Prepare blocking buffer for Western blot.

Protocol:
Add 12mL TBST. Add 0.6g non-fat milk. Vortex to mix. Add 2μL antibody. Resuspend the solution.
**11. Preparation of 1x TBST for Western blot**
Date: 01/08/2025
Aim:
Prepare 1x TBST for Western blot.
Protocol:
Add 100mL 10x TBST. Add 900mL ddH2O. Add 1mL Tween 20 (Pipette very slowly).
**12. Testing the effect of sulfate and sulfite on BL21-pET11a growth**
Date: 01/08/2025
Aim:
Testing the effect of different concentrations of sulfate and sulfite on BL21-pET11a growth.
Protocol:
Prepare LB broth with sulfate, LB broth with sulfite in different concentrations and LB broth(control). Take tubes prepared on 31/7 out of the incubator. Pipette 30uL LB with cultures out from tubes, add them into the LB broth(control), LB broth with different concentrations of sulfate and sulfite respectively. Measure OD600 every hour for 5 hours.

![](https://static.igem.wiki/teams/5685/julynotebook/fig-11-9.webp)

Figure 11.9 Bacteria cultured with sulfate and sulfite before checking the OD600 values

![](https://static.igem.wiki/teams/5685/julynotebook/fig-11-10.webp)

Figure 11.10 Using a spectrophotometer to check OD600 values

Results:

Table 11.3 Sulfite group, OD600 values of BL21-pET11a with different concentrations over 5 hours.

![](https://static.igem.wiki/teams/5685/julynotebook/tab-11-3.webp)
![](https://static.igem.wiki/teams/5685/julynotebook/fig-11-11.webp)

Figure 11.11 Sulfite group, curves (OD vs. time) for BL21-pET11a growth with different concentrations of sulfate added

Table 11.4 Sulfate group, OD600 values of BL21-pET11a with different concentrations over 5 hours

![](https://static.igem.wiki/teams/5685/julynotebook/table-11-4.webp)
![](https://static.igem.wiki/teams/5685/julynotebook/fig-11-12.webp)

Figure 11.12 Sulfate group, curves (OD vs. time) for BL21-pET11a growth with different concentrations of sulfate added

Conclusion:
Concentrations of sulfite and sulfate (0.01mM, 0.1mM and 1mM) do not affect the growth of bacteria BL21-pET11a.

#### Week 12

---

**1. Preparation of flushing test and bacterial growth test**
Date: 04/08/2025
Aim:
Shaking [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [3]pET-T7-INP-YFP(K523013)-csgA, [7]pET-T7-INP-YFP (K523013)-T7 tag, and BL21-pET11a stock for flushing test and bacterial growth test.
**2. Testing the effect of sulfuric acid on BL21-pET11a growth**
Date: 05/08/2025
Aim: Testing the effect of different concentrations of sulfuric acid on BL21-pET11a growth.
Protocol:
Prepare LB broth with sulfuric acid in different concentrations and LB broth(control). Take tubes prepared on4/7 out of the incubator. Pipette 30uL LB with cultures out from tubes, add them into the LB broth(control), LB broth with different concentrations of sulfuric acid respectively. Measure OD600 every hour for 5 hours.
Results:

Table 12.1. Sulfuric acid group, OD600 values of BL21-pET11a with different concentrations over 5 hours.

![](https://static.igem.wiki/teams/5685/augustlogbook/sulfuric-acid-growth-test.webp)
![](https://static.igem.wiki/teams/5685/augustlogbook/sulfuric-acid-result-graph.webp)

Figure 12.1. Sulfuric acid group, curves (OD vs. time) for BL21-pET11a growth with different concentrations of sulfate added.

Conclusion:
Concentrations of sulfuric acid (0.01mM, 0.1mM) do not affect the growth of bacteria BL21-pET11a.
**3. Flushing test for bacteria adhesion**
Date: 05/08/2025
Aim:
To verify the E.coli which carry the csgA gene resist rinsing with solutions, creating adhesion between the building surface.

Protocol:
4th time E.coli flushing test for adhesion using H2SO4, PBS and NaOH solutions of different pH values to flush the slide adhere with bacterial solution. Photograph with fluorescence microscope for subsequent colony counts with imageJ.
Results:
Bacteria [1]-BL21-INP-silicatein-INP-YFP-csgA, [3]-BL21-INP-YFP-csgA, [7]-pET-T7-INP-YFP-T7 tagsuccessfully adhere to the slide surface.

![](https://static.igem.wiki/teams/5685/augustlogbook/39.webp)

Figure 12.2. DIC and YFP images of [3]-BL21-INP-YFP-csgA without flushing (100x)

![](https://static.igem.wiki/teams/5685/augustlogbook/40.webp)

Figure 12.3. DIC and YFP images of [3]-BL21-INP-YFP-csgA flushed with pH3 H2SO4 (100x)

Conclusion: A significant number of bacteria retain on the slide surface even after flushing with solution across a range of pH conditions. This result is consistent across experimental bacteria: [1]-BL21-INP-silicatein-INP-YFP-csgA, [7]-pET-T7-INP-YFP-T7 tag. Also, low pH enhances adhesion of protein binding domains (CsgA).
**4. Preparation for qPCR of engineered E. Coli.**
Date: 06/08/2025
Aim:
To prepare for qPCR of engineered E. Coli. below:

* [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csg
* [2]pET-J23119-INP-silicatein
* [3]pET-T7-INP-YFP(K523013)-csgA
* [4]pET-J23119-ydeD (K4171005)
* [5]pET-J23119-yhaM
* [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta
* BL21-pET11a stock

Protocol:
Take 1 ml microbial whose OD value is between 0.5-0.6. Centrifuge the EP tube for 3 minutes at 10000 rpm, Wash twice with ddH2O. Centrifuge(10k rpm, 2.5 min) per washing. Remove medium. Add 1 mL TRIzol per well. Pipette to wash the well. Transfer the mixture into a new 1.5 mL EP tube. Incubation: 5 mins at room temp. Add 200 μL chloroform to allow protein to solidify and ppt. Vortex to mix. \*Conduct in fume hood\*. Incubation: 3 mins at room temp. Centrifuge: 12000 xg, 15 mins, 4°C. Transfer the aqueous layer to a new 1.5 mL EP tube. Add 0.5 mL 100% isopropanol to the aqueous layer to remove water. Incubation: 10 mins at room temp. Centrifuge: 12000 xg, 10 mins, 4°C. Remove the supernatant and wash the RNA pellet with 1 mL of 75% ethanol. Invert sample. Centrifuge: 14000 rpm, 5 mins, 4°C. Discard supernatant and wash the RNA pellet with 1 mL of 80% ethanol. Invert the sample. Repeat steps 17~18. Quick centrifuge: 14000 rpm, 5 mins, 4°C. Discard the supernatant. Dry the pellet for 10~20 mins at room temp. Resuspend RNA pellet with 30~60 μL RNase-free water(ddH2O). Incubation: 55°C, 10 mins. Store at -80°C. (Check concentration: use nanodrop on.)

![](https://static.igem.wiki/teams/5685/augustlogbook/41.webp)

Figure 12.4. Adding Trizol

**5. qPCR of engineered E. Coli.**
Date: 07/08/2025
Aim:
To do qPCR of engineered E. Coli. below:

* [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csg
* [2]pET-J23119-INP-silicatein
* [3]pET-T7-INP-YFP(K523013)-csgA
* [4]pET-J23119-ydeD (K4171005)
* [5]pET-J23119-yhaM
* [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta
* BL21-pET11a stock

Protocol:
Add 10 μL Fast SYBR Green PCR Master Mix to each well. Add 0.2 μL forward and reverse primers to each well. Add 1 ng (1 μL) corresponding cDNA into the wells. (Regarding the wells for H2O, add 1 μL ddH2O; regarding that of PET11a, add 1 ng PET11a empty vector.) Add 8.6 μL ddH2O to each well. Set up the instrument as follows:

Type of experiment: Quantitation - Comparative Cr(ΔΔCr)
Reagent: SYBR Green Reagent
Ramp speed: 2 hrs

**6. Preparation of sulfite reduction test for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**
Date: 05/08/2025
Aim:
Shaking BL21-pET11a and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta stock.
**7. Preparation of sulfite reduction test for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**
Date: 06/08/2025
Aim:
Culture [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta and BL21-pET11a with 0.1mM sodium sulfite and sodium sulfate for sulfite reduction test.

Protocol:
Prepare LB broth with 1mM sulfate, LB broth with sulfite in different concentration and LB broth(control). Take tubes prepared on 05/08 out of the incubator. Pipette 30uL LB with cultures out from tubes, add them into the LB broth(control), LB broth with different concentrations of sulfate and sulfite respectively. Incubate them in an incubator overnight.
**8. Sulfite reduction test for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**
Date: 07/08/2025
Aim:
Test whether [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta is capable of reducing sulfite to cysteine.

Protocol:
The test was carried out with Sangon cysteine content assay kit. By testing standard cysteine solution with concentrations of 2, 1, 0.5, 0.25, 0.125, 0.0625 mM, we obtained the standardised best-fit of different concentrations of cysteine by testing its absorbance at 600nm with the reagents in the Sangon cysteine content assay kit. Pipette 0.2mL of the incubated bacteria and 0.3mL of the Cysteine extract liquid. Centrifuge at 11000 rpm at 4°C for 10 minutes. Remove the supernatant. Preheat the spectrophotometer (600 nm) for 30 minutes.

Table 12.2. operation table for cysteine assay kit

![](https://static.igem.wiki/teams/5685/augustlogbook/2025-09-28-173142.webp)

![](https://static.igem.wiki/teams/5685/augustlogbook/42.webp)

Figure 12.5. BL21-pET11a control group sample after adding kit solutions, and waited for 15 minutes

Result:

Table 12.3. Data and cysteine concentration after adding 0.1mM sulfite in bacterial broth for 24 hours.

![](https://static.igem.wiki/teams/5685/augustlogbook/2025-09-28-173454.webp)

![](https://static.igem.wiki/teams/5685/augustlogbook/43.webp)

Figure 12.6. Cysteine concentration after adding 0.1mM sodium sulfite for 24 hours

Conclusion:
The concentration of cysteine in BL21-pET11a broth was higher than that of [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta broth, which did not meet the result we expected. We then realized that it was because [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta directly degrades sulfite into hydrogen sulfide instead of cysteine. We decided to use a hydrogen sulfide kit for the experiment after that.
**9. Preparation of H2S formation pre-test of [5]pET-J23119-yhaM**
Date: 07/08/2025
Aim:
Prepare for the H2S formation pre-test of [5]pET-J23119-yhaM

Protocol:
With final concentrations of 2.862mM Ampicillin and 4mM of cysteine in LB broth, the 4mM-Cysteine-LB-Amp broth is prepared. With final concentrations of 2.862mM Ampicillin and 8mM of cysteine in LB broth, the 8mM-Cysteine-LB-Amp broth is prepared. With final concentrations of 2.862mM Ampicillin and 8mM of cysteine in LB broth, the LB-Amp broth is prepared. Add 30μL (1:100 v:v) of bacteria stock (BL21-pET11a) to 3mL of 3 broths each. Then incubate them at 37°C 220rpm overnight.
**10. H2S formation pre-test of [5]pET-J23119-yhaM**
Date: 08/08/2025
Aim:
To test the appropriate range of Cysteine added to the bacteria before incubating.

Protocol:
We use the Boxbio H2S content assay kit to test the H2S amount in the cultured bacteria. Add 100μL of cultured bacteria to the extract liquid. Sonify and centrifuge it. Test the supernatants as the samples. After adding the reagents, light-protected colour development stands for 15 minutes. Test the absorbance at 680 nm.

Table 12.4. operation table of H2S content assay kit

![](https://static.igem.wiki/teams/5685/augustlogbook/2025-09-28-181946.webp)

Result:

![](https://static.igem.wiki/teams/5685/augustlogbook/44.webp)

Figure 12.7. Colour of samples after light-protected colour development (From Left To Right: 8mM pre-added Cysteine, 4mM pre-added cysteine, no cysteine added)

Table 12.4. Absorbance at 680 nm of samples testing with H2S content assay kit

![](https://static.igem.wiki/teams/5685/augustlogbook/2025-09-28-182404.webp)

Conclusion:
Adding 8mM of Cysteine has the best effect. We used this as the concentration of Cysteine in the H2S formation tests afterwards.

#### Week 13

---

**1. Preparation of Qpcr, flushing test ,[6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta reducing sulfate sulfite test**
Date: 11/08/2025
Aim:
Shaking[1]-[8] and BL21-pET11a, details of plasmids as follwings:
[1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA
[2] pET-J23119-INP-silicatein
[3] pET-T7-INP-YFP(K523013)-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-silicatein (K1890001)-T7 tag
BL21-pET11a stock

Protocol:
**[1]&[2]&[4]&[5]&pET11a**
Measure 4mL LB broth, pipette 4mL of LB broth and 4 uL of Ampicillin into each Polystyrene Round-Bottom Tube. Place 40uL [1]-[8] & pET11a into each tube. Place onto the incubator (at 250 rpm and 37C) overnight.
**[3]&[6]&[7]&[8]**
Measure 4mL LB broth, pipette 4mL of LB broth and 4 uL of Ampicillin into each Polystyrene Round-Bottom Tube.Place 40uL [1]-[8] & pET11a into each tube. When OD600=0.6, Add IPTG induce, incubate at 250 rpm and 25 °C overnight.
**2. Qpcr for all engineered E. Coli.**
Date: 12/08/2025
Aim:
qPCR for plasmids(all plasmids and control group) below:

* [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csg
* [2]pET-J23119-INP-silicatein
* [3]pET-T7-INP-YFP(K523013)-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-silicatein (K1890001)-T7 tag
* BL21-pET11a stock

Protocol:
Take 8 ml microbial whose OD value is between 0.5-0.6. Centrifuge the EP tube for 5 minutes at 4000 xg. Add 1 mL TRIzol per well. resuspend until no precipitate is seen. Transfer the mixture into a new 1.5 mL EP tube. Incubation: 10 mins at room temp. Centrifuge: 12000 rpm for 10 mins at 4C Transfer supernatant to new ep tube. \*big tip THEN small tip when using Pipette Add 200 μL chloroform. Vortex to mix. \*Conduct in fume hood\* Incubation: 3 mins at room temp. Centrifuge: 12000 rpm, 10 mins, 4°C. Transfer the aqueous layer to a new 1.5 mL EP tube. \*big tip then small tip Add 0.6 mL 100% isopropanol to the aqueous layer. Incubation: 20 mins at room temp. Centrifuge: 12000 rpm, 10 mins, 4°C. Remove the supernatant and wash the RNA pellet(wash out isopropanol to minimise its effect) with 1 mL of 75% ethanol. Store at -20°C in 75% ethanol. (Check concentration: use nanodrop one)
Take the samples out of -20C. Centrifuge at 12000rpm for 5 minutes. Remove the supernatant carefully. Place the tubes horizontally to allow the pellet to dry. Check concentration. Resuspend 5x iScript Reaction Mix until no precipitate is seen. Add the following on ice. Resuspend thoroughly.
**3. Sulfite reduction test for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**
Date: 11/08/2025
Aim:
Shaking[1]-[8] and BL21-pET11a, details of plasmids as follwings:

* [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csg
* [2]pET-J23119-INP-silicatein
* [3]pET-T7-INP-YFP(K523013)-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-silicatein (K1890001)-T7 tag
* BL21-pET11a stock

Protocol:
**[1]&[2]&[4]&[5]&pET11a**
Measure 4mL LB broth, pipette 4mL of LB broth and 4 uL of Ampicillin into each Polystyrene Round-Bottom Tube. Place 40uL [1]-[8] & pET11a into each tube. Place onto the incubator (at 250 rpm and 37C) overnight.
**[3]&[6]&[7]&[8]**
Measure 4mL LB broth, pipette 4mL of LB broth and 4 uL of Ampicillin into each Polystyrene Round-Bottom Tube.Place 40uL [1]-[8] & pET11a into each tube. When OD600=0.6, Add IPTG induce, incubate at 250 rpm and 25 °C overnight.
**4. Preparation of H2S formation test for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**
Date: 12/08/2025
Aim:
Prepare for the H2S formation pre-test of [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta

Protocol:
With final concentrations of 0.05mM Ampicillin and 0.1mM of sulfite in LB broth. Add 30μL (1:100 v:v) of bacteria stock (BL21-pET11a and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta) to 3mL of 2 broths. Then incubate them at 37°C 220rpm overnight.
**5. Hydrogen sulfide formation test of [6]pET-T7-RBS-NSP4-sulfite reductase alpha-sulfite reductase beta**
Date: 13/08/2025 and 14/08/2025
Aim:
To test whether [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta is capable of reducing sulfite to H2S.

Protocol:
**[1]&[2]&[4]&[5]&pET11a**
We use the Boxbio H2S content assay kit to test the H2S amount in the cultured bacteria. Add 100μL of cultured bacteria to the extract liquid. Sonify and centrifuge it. Test the supernatants as the samples. After adding the reagents, light-protected colour development stands for 15 minutes. Test the absorbance at 680 nm.

Table 13.1. operation table of H2S content assay kit

![](https://static.igem.wiki/teams/5685/augustlogbook/2025-09-28-181946.webp)

![](https://static.igem.wiki/teams/5685/augustlogbook/45.webp)

Figure 13.1. Adding hydrogen sulfide assay kit solutions into samples.

![](https://static.igem.wiki/teams/5685/augustlogbook/46.webp)

Figure 13.2. Testing absorbance at 680nm for samples.

Result:

Table 13.2. Data and the H2S concentration of each bacterial broth after adding sodium sulfate, sodium sulfite and sulfuric acid for 24 hours (13/08/2025)

![](https://static.igem.wiki/teams/5685/augustlogbook/2025-09-28-184320.webp)

Table 13.3. Data and the H2S concentration of each bacterial broth after adding sodium sulfate, sodium sulfite and sulfuric acid for 24 hours (14/08/2025)

![](https://static.igem.wiki/teams/5685/augustlogbook/2025-09-28-184516.webp)

![](https://static.igem.wiki/teams/5685/augustlogbook/47.webp)

Figure 13.3. H2S concentration of each bacterial broth after adding sodium sulfate, sodium sulfite and sulfuric acid for **24hours**

![](https://static.igem.wiki/teams/5685/augustlogbook/48.webp)

Figure 13.4. H2S concentration of each bacterial broth after adding sodium sulfate, sodium sulfite and sulfuric acid for **48hours**

Conclusion:
The concentration results of BL21-pET11a group and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta on both 13/08/2025 and 14/08/2025 were at a similar level. It was probably because the amount of hydrogen sulfide produced was little. Also, hydrogen sulfide is a gas, and a majority of it might spread away during the experiments. We decided to use a Megazyme total sulfite assay kit to directly determine the capability of [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta reducing sulfite later on.
**6. Preparation of H2S formation test for [5]pET-J23119-yhaM**
Date: 13/08/2025
Aim:
Test whether the YhaM gene reduces cysteine into H2S.

Protocol:
With final concentrations of 2.862mM Ampicillin and 8mM of cysteine in LB broth, the 8mM-Cysteine-LB-Amp broth is prepared. With final concentrations of 2.862mM Ampicillin and 8mM of cysteine in LB broth, the LB-Amp broth is prepared. Add 30μL (1:100 v:v) of bacteria stock (BL21-pET11a and [5]pET-J23119-yhaM) to 3mL of 2 broths to 3 of each. Then incubate them at 37°C 220rpm overnight.
Result:

![](https://static.igem.wiki/teams/5685/augustlogbook/49.webp)

Figure 13.5. H2S amount of [5]pET-J23119-yhaM and BL21-pET11a after incubating with 8mM cysteine overnight

Conclusion:
The test verifies that [5]pET-J23119-yhaM can reduce cysteine into H2S because it has more H2S content after incubating with 8mM cysteine comparing with control group (BL21-pET11a).
**7. Flushing test (6th)**
Date: 12/08/2025
Aim:
To verify the E.coli which carry the csgA gene resist rinsing with solutions, creating adhesion between the building surface.

Protocol:
1st, 2nd and 3rd time E.coli flushing test for adhesion using H2SO4, PBS and NaOH solutions of different pH values to flush the slide adhere with bacterial solution. Photograph with fluorescence microscope for subsequent colony counts with imageJ.
Result:

![](https://static.igem.wiki/teams/5685/augustlogbook/50.webp)

Figure 13.6. DIC and YFP images of BL21-INP-silicatein-INP-YFP-csgA without flushing (100x)

![](https://static.igem.wiki/teams/5685/augustlogbook/51.webp)

Figure 13.7. DIC and YFP images of BL21-INP-silicatein-INP-YFP-csgA flushed with pH3 H2SO4 (100x)

![](https://static.igem.wiki/teams/5685/augustlogbook/52.webp)

Figure 13.8. DIC and YFP images of BL21-T7-INP-YFP-csgA without flushing (100x)

![](https://static.igem.wiki/teams/5685/augustlogbook/53.webp)

Figure 13.9. DIC and YFP images of BL21-T7-INP-YFP-csgA flushed with pH3 H2SO4 (100x)

Conclusion:
This demonstrates that csgA enhances the adhesion of the protein-binding domain to glass slides at low pH, while the YFP design enables observation of bacterial colonies under a microscope. The INP directs CsgA to the cell surface or periplasm, enabling bacterial adhesion to building surfaces.
**8. Preparation of sulfite reduction test with cysteine added for [4]pET-J23119-ydeD (K4171005) and [5]pET-J23119-yhaM**
Date: 15/08/2025
Aim:
Shaking BL21-pET11a, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM stock.
**9. Preparation of sulfite reduction test for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**
Date: 15/08/2025
Aim:
Shaking BL21-pET11a and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta stock.

Protocol:
With final concentrations of 0.05mM Ampicillin and 0.1mM of sulfite in LB broth. Add 30μL (1:100 v:v) of bacteria stock (BL21-pET11a and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta) to 3mL of 2 broths. Then incubate them at 37°C 220rpm overnight.
**10. Preparation of sulfite reduction test with cysteine added for [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**
Date: 16/08/2025
Aim:
Prepare for the sulfite reduction test.

Protocol:
**Control Group:**

Culture BL21-pET11a in 5mL LB + 5μL Amp in a tube.
Culture [4]pET-J23119-ydeD (K4171005) in 5mL LB + 5μL Amp in a tube.
Culture [5]pET-J23119-yhaM in 5mL LB + 5μL Amp in a tube.
Culture [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta
Incubate at 37°C, 220 rpm
**Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Add IPTG when the OD600 value reaches 0.6
Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Move to IPTG 37°C, 220 rpm after adding IPTG.**
Incubate them overnight

**Sulfite Group:**

Add final concentrations of 1mM sodium sulfite to the Amp-LB broth.
Culture BL21-pET11a in 5mL of the broth made in Step 1 in a tube.
Culture [4]pET-J23119-ydeD (K4171005) in 5mL of the broth made in Step 1 in a tube.
Culture [5]pET-J23119-yhaM in 5mL of the broth made in Step 1 in a tube.
Culture [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta of the broth made in Step 1.
Incubate at 37°C, 220 rpm
**Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Add IPTG when the OD600 value reaches 0.6**
**Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Move to IPTG 37°C, 220 rpm after adding IPTG.**
Incubate them overnight

![](https://static.igem.wiki/teams/5685/augustlogbook/54.webp)

Figure 13.10. Incubating bacterial culture after adding 1mM sodium sulfite and 1mM cysteine.

#### Week 14

---

**1. Sulfite reduction test with cysteine added for [4]pET-J23119-ydeD (K4171005) and [5]pET-J23119-yhaM**
Date: 18/08/2025
Aim:
To test whether [4]pET-J23119-ydeD (K4171005) and [5]pET-J23119-yhaM reduce sulfite.

Protocol:
Pipette 50uL bacteria solution to 1.5mL tube. Add 50uL cold 1M perchloric acid, mix thoroughly. Centrifuge at 1500g for 10 mins. Pipette 50uL supernatant fluid into a new 1.5mL tube. Add 25uL 1M NaOH into the new tube with supernatant fluid, mix thoroughly. Pipette 1.25mL distilled water, 250uL solution 1 (buffer), 50uL sample solution(prepared in step 1~5), 10uL solution 3 and 100uL solution 2 (NADH) into a 15mL tube, mix them well. Wait for 4 mins. Set spectrophotometer to 340nm, pipette 1mL solution into a curvette, record the OD. Pipette the solution back to the tube from the cuvette, use ddH2O, flush the cuvette for 3 times. Pipette 10uL suspension 4 (sulfite oxidase) and mix thoroughly after testing the OD, wait for 30 mins. Set spectrophotometer to 340nm, pipette 1mL solution into a cuvette, record the absorbance.

![](https://static.igem.wiki/teams/5685/augustlogbook/15-2.webp)

Figure 14.1. Checking absorbance at 340nm for samples

![](https://static.igem.wiki/teams/5685/augustlogbook/15-3.webp)

Figure 14.2. Materials needed for absorbance at 340nm

Result:

Table 14.1. Data and sulfite concentration of samples

![](https://static.igem.wiki/teams/5685/augustlogbook/2025-09-28-205115.webp)

![](https://static.igem.wiki/teams/5685/augustlogbook/15-4.webp)

Figure 14.3. Sulfite concentration after adding 1mM sodium sulfite and 1mM cysteine for 48 hours

Conclusion:
All experimental groups showed a decrease in sulfite concentration. However, extra cysteine should not be added since there was cysteine inside the bacterial system already, therefore we decided to conduct experiments without adding extra cysteine in LB broth in the following weeks.
**2. Sulfite reduction test with cysteine added for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**
Date: 18/08/2025
Aim:
To test whether [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta reduce sulfite reduces sulfite.

Protocol:
Pipette 50uL bacteria solution to 1.5mL tube. Add 50uL cold 1M perchloric acid, mix thoroughly. Centrifuge at 1500g for 10 mins. Pipette 50uL supernatant fluid into a new 1.5mL tube. Add 25uL 1M NaOH into the new tube with supernatant fluid, mix thoroughly. Pipette 1.25mL distilled water, 250uL solution 1 (buffer), 50uL sample solution(prepared in step 1~5), 10uL solution 3 and 100uL solution 2 (NADH) into a 15mL tube, mix them well. Wait for 4 mins. Set spectrophotometer to 340nm, pipette 1mL solution into a curvette, record the OD. Pipette the solution back to the tube from the cuvette, use ddH2O, flush the cuvette for 3 times. Pipette 10uL suspension 4 (sulfite oxidase) and mix thoroughly after testing the OD, wait for 30 mins. Set spectrophotometer to 340nm, pipette 1mL solution into a cuvette, record the absorbance.
Result:
The concentration of sulfite of each experimental group was at a similar level, so we decided to conduct the experiment again later in this week.
**3. Preparation of sulfite reduction test with cysteine added for [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**
Date: 18/08/2025
Aim:
Shaking BL21-pET11a, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta stock.
**4. Preparation of sulfite reduction test for [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**
Date: 19/08/2025
Aim:
Prepare for the sulfite reduction test.

Protocol:
**Control Group:**

Culture BL21-pET11a in 5mL LB + 5μL Amp in a tube.
Culture [4]pET-J23119-ydeD (K4171005) in 5mL LB + 5μL Amp in a tube.
Culture [5]pET-J23119-yhaM in 5mL LB + 5μL Amp in a tube.
Culture [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta
Incubate at 37°C, 220 rpm
**Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Add IPTG when the OD600 value reaches 0.6
Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Move to IPTG 37°C, 220 rpm after adding IPTG.**
Incubate them overnight

**Sulfite Group:**

Add final concentrations of 1mM sodium sulfite to the Amp-LB broth.
Culture BL21-pET11a in 5mL of the broth made in Step 1 in a tube.
Culture [4]pET-J23119-ydeD (K4171005) in 5mL of the broth made in Step 1 in a tube.
Culture [5]pET-J23119-yhaM in 5mL of the broth made in Step 1 in a tube.
Culture [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta of the broth made in Step 1.
Incubate at 37°C, 220 rpm
**Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Add IPTG when the OD600 value reaches 0.6**
**Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Move to IPTG 37°C, 220 rpm after adding IPTG.**
Incubate them overnight

**5. Sulfite reduction test for [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**
Date: 18/08/2025
Aim:
To test whether [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta are capable of reducing sulfite.

Protocol:
Pipette 50uL bacteria solution to 1.5mL tube. Add 50uL cold 1M perchloric acid, mix thoroughly. Centrifuge at 1500g for 10 mins. Pipette 50uL supernatant fluid into a new 1.5mL tube. Add 25uL 1M NaOH into the new tube with supernatant fluid, mix thoroughly. Pipette 1.25mL distilled water, 250uL solution 1 (buffer), 50uL sample solution(prepared in step 1~5), 10uL solution 3 and 100uL solution 2 (NADH) into a 15mL tube, mix them well. Wait for 4 mins. Set spectrophotometer to 340nm, pipette 1mL solution into a curvette, record the OD. Pipette the solution back to the tube from the cuvette, use ddH2O, flush the cuvette for 3 times. Pipette 10uL suspension 4 (sulfite oxidase) and mix thoroughly after testing the OD, wait for 30 mins. Set spectrophotometer to 340nm, pipette 1mL solution into a cuvette, record the absorbance.

![](https://static.igem.wiki/teams/5685/augustlogbook/15-6.webp)

Figure 14.4. Checking absorbance at 340 for sample.

Result:
The sulfite concentration of [4]ydeD was higher than that of [5]yhaM, [6]sulfite reductase and BL21-pET11a, and the concentration of BL21-pET11a was the lowest, which was not normal. It might be due to the slight time error of adding solutions and the opportunities of sulfite oxidizing into sulphur dioxide.
**6. qPCR for all engineered E. Coli.**
Date: 19/08/2025
Aim:
qPCR for plasmids(all plasmids and control group) below:

* [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csg
* [2]pET-J23119-INP-silicatein
* [3]pET-T7-INP-YFP(K523013)-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-silicatein (K1890001)-T7 tag
* BL21-pET11a stock

Protocol:
Add 10 μL Fast SYBR Green PCR Master Mix to each well. Add 0.2 μL forward and reverse corresponding primers to experimental wells. Add 0.2 μL forward and reverse control primers ([4] for [1][2][3]; [1] for [4] [5]; [5] for [6][7][8]) to control wells. Add 0.2 μL forward and reverse 16s RNA primer to internal reference wells. Add 1 ng (1 μL) corresponding cDNA into the wells. (Regarding the wells for H2O, add 1 μL ddH2O; regarding that of PET11a, add 1 ng PET11a empty vector.) Add 8.6 μL ddH2O to each well.
Set up the instrument as follows:

* Type of experiment: Quantitation - Comparative Cr(ΔΔCr)
* Reagent: SYBR Green Reagent
* Ramp speed: 2 hrs

Then run.
Result:

![](https://static.igem.wiki/teams/5685/augustlogbook/15-1.webp)

Figure 14.5. qPCR result of all our engineered E. Coli.

Conclusion:
The test verifies that our engineered E.Coli. carries out out target protein.
**7. Silica formation test on limestones**
Date: 19/08/2025
Aim:
Testinjg whether silca forms on limestones to narrow the cracks.

Protocol:
We soaked
Then run.
Result:
See [Results: Repairing](https://2025.igem.wiki/puiching-macau/results)

Conclusion:
The test verifies that [1]INP-silicatein + INP-YFP-csgA narrows the cracks on limestones significantly.

#### Week 15

---

**1. Preparation of silica formation test for [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag**
Date: 28/08/2025

Aim:
Prepare for the silica formation test of [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag.

Protocol:
A volume of 5 mL of LB broth was measured. Then, 5 mL of LB broth and 5 μL of ampicillin were pipetted into each polystyrene round-bottom tube. Next, 50 μL of each construct—[1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2] pET-J23119-INP-silicatein, and [8] pET-T7-RBS-silicatein (K1890001)-T7 tag—along with BL21-pET-11a, were added to their respective tubes. The tubes were placed in an incubator and shaken overnight at 250 rpm and 37°C.

**Only for [8]pET-T7-RBS-silicatein (K1890001)-T7 tag: When OD600=0.6, added IPTG to induce protein production. Then incubated at 250 rpm and 25 °C overnight.**
**Preparation of Reagents:**

* 5% m/v oxalic acid solution
* 0.04 M ascorbic acid solution
* 1% v/v glycerol solution
* 100 g/L acidified ammonium molybdate solution
* 0.17 M HCl
* Hydrolyzed TEOS stock solution
* Silicon stock solution

**Preparation of the 4mM TEOS Solution:**
24 hours after the initial hydrolysis of TEOS (the time required for complete hydrolysis), transfer 0.107 mL of the hydrolyzed TEOS into 95.593 mL of water.
**Preparation of 5% HF Solution:**
Prepare 5% HF solution inside a fume hood by diluting 5.2 g of 48% wt HF into 50 mL of water. Use only plastic equipment for the entire procedure.

**2. Silica formation test for [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag**
Date: 29/08/2025
Aim:
To test whether [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag form silica.

Protocol:
**Biomineralization:**
4 groups of cultured bacteria are spun down at 4000 rpm for 10 mins. Supernatants are removed. The cell pellets are resuspended in 5ml of water, then transferred to a 125ml flask and marked to 25ml with water. 6ml of 4mM TEOS is added to each bacteria type. Incubate for three nights at 250 rpm and 37C.
**Plotting the calibration curve of silicon concentration and optical density:**
Transfer 920ul of different concentration of silicon solution (0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/L) and 920ul of water into 12 EP tubes. Add 10ul of acidified ammonium molybdate solution, 50ul of oxalic acid solution, 10ul of ascorbic acid solution, and 10ul of glycerin solution in this order. 20 mins later, the 12 samples are transferred into 12 cuvettes and scanned under 825nm using a spectrophotometer three times. Average absorbances are acquired.

![](https://static.igem.wiki/teams/5685/augustlogbook/2025-09-28-213942.webp)

Figure 15.1. Standard solutions with known concentration ready for spectrophotometer scanning

![](https://static.igem.wiki/teams/5685/newphoto/figure-15-2-n1.webp)

Figure 15.2. Checking absorbance at 825nm of samples

Result:

Table 15.1. Silicon concentrations and absorbance at 825nm of samples.

![](https://static.igem.wiki/teams/5685/augustlogbook/2025-09-28-214254.webp)

![](https://static.igem.wiki/teams/5685/augustlogbook/16-3.webp)

Figure 15.3. The calibration curve between OD value and silicon concentration

Comclusion:
The test is successful and shows a linear relationship between silicon concentration and OD values. The data acquired are expected and can be represented by a linear equation.
**3. qPCR for sulfate reduction pathway enzymes**
Date: 29/08/2025
Aim:
To test whether the amount of sulfate reduction pathway enzymes increases to determine whether the pathway is sped up.

Protocol:
Add 10 μL Fast SYBR Green PCR Master Mix to each well. Add 0.2 μL forward and reverse corresponding primers to experimental wells. Add 0.2 μL forward and reverse control primers ([4] for [1][3]; [1] for [5]) to control wells. Add 0.2 μL forward and reverse 16s RNA primer to internal reference wells. Add 1 ng (1 μL) corresponding cDNA into the wells. (Regarding the wells for H2O, add 1 μL ddH2O; regarding that of PET11a, add 1 ng PET11a empty vector.) Add 8.6 μL ddH2O to each well. Set up the instrument as follows:

* Type of experiment: Quantitation - Comparative Cr(ΔΔCr)
* Reagent: SYBR Green Reagent
* Ramp speed: 2 hrs

Result:

![](https://static.igem.wiki/teams/5685/augustlogbook/16-4.webp)

Figure 15.4. Result of qPCR of sulfate reduction pathway enzymes

Conclusion:
The expression of all enzymes in the sulfate reduction pathway of [4]ydeD gene are obviously higher than both control and [5]yhaM gene, while [5]yhaM gene expresses better in CysC protein when compared to the control group, validating that our plasmids are capable of reducing sulfate effectively.

#### Week 16

---

**1. Silica formation test for [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag**
Date: 01/09/2025

Aim:
To test whether [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag form silica.

Protocol:
Always for this procedure, volume of sample:volume of acidified ammonium molybdate:volume of oxalic acid:volume of glycerin solution:volume of ascorbic acid=92:1:5:1:1.
After incubation, the bacteria are centrifuged at 4500 rpm for 20 minutes. The supernatants are collected and further centrifuged at 5000 rpm for 200 minutes. The supernatants from the second centrifugation are removed, and the precipitates are left to dry. The samples are resuspended in 1 ml of 0.02 M NaOH, and 1 M HCl is added to adjust the pH to 1.6-1.9. Acidified ammonium molybdate is added to the dissolved samples. Five minutes later, oxalic acid, ascorbic acid, and glycerin solutions are added one after the other. The samples are left for 20 minutes for blue color development. Each sample is diluted 10-fold, and the OD values are measured at 825 nm. The mass of the precipitate can be determined by referring to the corresponding concentration. The experiment is conducted twice.

![](https://static.igem.wiki/teams/5685/silica/20251001185736.webp)

Figure 16.1. Adjusting sample pH values with HCl

![](https://static.igem.wiki/teams/5685/silica/20251001185749.webp)

Figure 16.2. Blue color formation across four samples

Results:

Table 16.1. Data acquired across 4 samples

![](https://static.igem.wiki/teams/5685/silica/20251001190059.webp)

![](https://static.igem.wiki/teams/5685/silica/data-4.webp)

Figure 16.3. OD values of 4 different samples

![](https://static.igem.wiki/teams/5685/silica/data-2.webp)

Figure 16.4. Mass of biosilica formed in 4 different samples

#### Week 17

---

**1. Preparation of sulfate reduction test with cysteine added for [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**

Date: 09/09/2025

Aim:
Shaking BL21-pET11a [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta stock.

**2. Preparation of sulfite and sulfate reduction test with cysteine added for [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**

Date: 10/09/2025

Protocol:

Control Group:
1.Culture BL21-pET11a in 5mL LB + 5μL Amp in a tube.
2.Culture [4]pET-J23119-ydeD (K4171005) in 5mL LB + 5μL Amp in a tube.
3.Culture [5]pET-J23119-yhaM in 5mL LB + 5μL Amp in a tube.
4.Culture [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta
5.Incubate at 37°C, 220 rpm
6.Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Add IPTG when the OD600 value reaches 0.6
7.Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Move to IPTG 37°C, 220 rpm after adding IPTG.
8.Incubate them overnight

Sulfite Group:
1.Add final concentrations of 1mM sodium sulfite to the Amp-LB broth.
2.Culture BL21-pET11a in 5mL of the broth made in Step 1 in a tube.
3.Culture [4]pET-J23119-ydeD (K4171005) in 5mL of the broth made in Step 1 in a tube.
4.Culture [5]pET-J23119-yhaM in 5mL of the broth made in Step 1 in a tube.
5.Culture [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta of the broth made in Step 1.
6.Incubate at 37°C, 220 rpm
7.Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Add IPTG when the OD600 value reaches 0.6
8.Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Move to IPTG 37°C, 220 rpm after adding IPTG.
9.Incubate them overnight.

Sulfate Group:
1.Add final concentrations of 1mM sulfuric acid to the Amp-LB broth.
2.Culture BL21-pET11a in 5mL of the broth made in Step 1 in a tube.
3.Culture [4]pET-J23119-ydeD (K4171005) in 5mL of the broth made in Step 1 in a tube.
4.Culture [5]pET-J23119-yhaM in 5mL of the broth made in Step 1 in a tube.
5.Culture [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta of the broth made in Step 1.
6.Incubate at 37°C, 220 rpm
7.Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Add IPTG when the OD600 value reaches 0.6
8.Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Move to IPTG 37°C, 220 rpm after adding IPTG.
9.Incubate them overnight.

**3. Sulfite reduction test at 0 hours and making sulfite concentration-Δabsorbance 340 nm standard curve for total sulfite assay kit**

Date: 10/09/2025

Aim:
Testing the accuracy of sulfite kit, and making a sulfite standard curve for the total sulfite assay kit.

Protocol:

Making sulfite standard solution with 0, 0.3, 0.6, 1. 1.3, 2 mM standard solution. Pipette 50uL bacteria solution to 1.5mL tube. Add 50uL cold 1M perchloric acid, mix thoroughly. Centrifuge at 1500g for 10 mins. Pipette 50uL supernatant fluid into a new 1.5mL tube. Add 25uL 1M NaOH into the new tube with supernatant fluid, mix thoroughly. 250uL solution 1 (buffer), 100uL solution 2(NADH), 5uL sample solution, 125uL distilled water, 10uL solution 3 into a well in the 96 well-flat bottom plate, mix them well. Wait for 4 mins. Set spectrophotometric multiwell plate reader to 340nm, record the OD. Pipette 10uL suspension 4 (sulfite oxidase) and mix thoroughly after testing the OD, wait for 30 mins. Set spectrophotometric multiwell plate reader to 340nm, record the absorbance.

![](https://static.igem.wiki/teams/5685/sepnotebook/fig-17-1.webp)

Figure 17.1. Adding kit solution to samples.

Results:

![](https://static.igem.wiki/teams/5685/sepnotebook/fig-17-2.webp)

Figure 17.2. Sulfite concentration-Δabsorbance 340nm standard curve

Conclusion:

The sulfite concentration in experimental groups (with 1mM sulfite added) were higher than that of control (except for BL21-pET11a), and we decided to conduct the experiment again using cuvette.

**4. Sulfate reduction test added for [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**

Date: 12/09/2025

Aim: To test whether [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta are capable of reducing sulfate.

Protocols:

For the standard group, add 20 uL of the 60 mM Standard to 580 uL of water to prepare a 2.0 mM Standard solution. Add 0, 50, 100, and 200 uL of the 2.0 mM Standard solution into tubes. Add water to each tube to bring the volume to 200uL, generating 0 (blank), 0.5, 1.0, and 2.0 mM standards. Add 100uL of TCA Reagent to 200 µL of each standard, and mix. Transfer 200 uL of each standard into separate wells of 96 well plates. For the bacterial samples, check the OD600 values and dilute them into similar levels. Pipette mix 200 µL of sample and 100 µL of TCA Reagent in a 1.5 mL microcentrifuge tube. Spin down protein precipitates 5 minutes at 14,000 rpm on a table centrifuge. Transfer 200 µL of supernatant into separate wells of a 96 well plate. Add 100 µL of the Master Reaction Mix to each of the appropriate wells preferably using a pipette and mix well. Incubate the reaction at room temperature for 5 minutes. Measure the optical density of the samples at 600 nm for assay.

Results:

Table 17.1. Data and sulfate concentrations after adding 1mM sulfuric acid for 48 hours.

![](https://static.igem.wiki/teams/5685/sepnotebook/fig-17-3.webp)
![](https://static.igem.wiki/teams/5685/sepnotebook/fig-17-4.webp)

Figure 17.3. Sulfate concentration of each sample after adding 1mM sulfuric acid for 48hours.

Conclusion:

The result of 1mM sulfate reduction test reached our expected result, in which the sulfate concentration of [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta broth were all lower than that of the BL21-pET11a broth. For this test, we used LB broth as a blank, after minusing the absorbance of blank, all of the concentrations reached a range to less than 1mM, which became normal. The result indicates that the sulfate reduction test succeeded, our plasmids are capable of reducing sulfate.

#### Week 18

---

**1. Preparation of silica formation test for [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag**
Date: 15/09/2025

Aim:
Prepare for the silica formation test of [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag.

Protocols:
Measure 5mL LB broth, pipette 5mL of LB broth and 5uL of ampicillin into each polystyrene round-bottom Tube. Place 50uL [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, [8]pET-T7-RBS-silicatein (K1890001)-T7 tag & BL21-pET-11a into each tube. Place onto the incubator (at 250 rpm and 37°C) overnight.
Only for [8]pET-T7-RBS-silicatein (K1890001)-T7 tag: When OD600=0.6, Add IPTG to induce protein production. Then incubate at 250 rpm and 25 °C overnight.

**2. Preparation of sulfite reduction test with cysteine added for [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**

Date: 16/09/2025

Aim:
Shaking BL21-pET11a [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta stock.

**3. Silica formation test for [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag**
Date: 16/09/2025

Aim:
To test whether [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag form silica using an improved method.

Protocols:
Cultured [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, [8]pET-T7-RBS-silicatein (K1890001)-T7 tag, and BL21-pET-11a are spun down at 4000 rpm for 10 mins. Supernatants are removed. The cell pellets are resuspended in 5ml of water, then transferred to a 125ml flask and marked to 25ml with water. 6ml of 4mM TEOS is added to each bacteria type. Incubate for three nights at 250 rpm and 37℃.

**4. Preparation of sulfite reduction test with cysteine added for [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**

Date: 17/09/2025

Aim:
Prepare for the sulfite reduction test.

Protocols:

Control Group:
1.Culture BL21-pET11a in 5mL LB + 5μL Amp in a tube.
2.Culture [4]pET-J23119-ydeD (K4171005) in 5mL LB + 5μL Amp in a tube.
3.Culture [5]pET-J23119-yhaM in 5mL LB + 5μL Amp in a tube.
4.Culture [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta
5.Incubate at 37°C, 220 rpm
6.Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Add IPTG when the OD600 value reaches 0.6
7.Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Move to IPTG 37°C, 220 rpm after adding IPTG.
8.Incubate them overnight.

Sulfite Group:
1.Add final concentrations of 1mM sodium sulfite to the Amp-LB broth.
2.Culture BL21-pET11a in 5mL of the broth made in Step 1 in a tube.
3.Culture [4]pET-J23119-ydeD (K4171005) in 5mL of the broth made in Step 1 in a tube.
4.Culture [5]pET-J23119-yhaM in 5mL of the broth made in Step 1 in a tube.
5.Culture [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta of the broth made in Step 1.
6.Incubate at 37°C, 220 rpm
7.Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Add IPTG when the OD600 value reaches 0.6
8.Only for [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta: Move to IPTG 37°C, 220 rpm after adding IPTG.
9.Incubate them overnight.

**5. Silica formation test for [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag**
Date: 17/09/2025

Aim:
To test whether [1]pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA, [2]pET-J23119-INP-silicatein, and [8]pET-T7-RBS-silicatein (K1890001)-T7 tag form silica using an improved method.

Protocols:
After incubation, the samples are centrifuged at 5000 rpm for 220 minutes. The supernatants are removed, and the precipitates are left to dry. Each sample is resuspended in 0.5 ml of water. Then, 90 µl of each sample is pipetted into an EP tube, repeated for all four samples. Acidified ammonium molybdate is added to the dissolved samples. Five minutes later, oxalic acid, ascorbic acid, and glycerin solutions are added one after the other. The samples are left for 20 minutes for blue color development. The samples are diluted 10-fold, and the OD values are measured at 825 nm. This group of samples is called the non-NaOH samples.
For the rest of the samples, 1 ml of 0.2 M NaOH is added to each sample. The samples are heated using a water bath for 7 minutes. Then, 1 M HCl is added to adjust the pH to 1.6-1.9. Acidified ammonium molybdate is added to the dissolved samples. Five minutes later, oxalic acid, ascorbic acid, and glycerin solutions are added one after the other. The samples are left for 20 minutes for blue color development. The samples are diluted 10-fold, and the OD values are measured at 825 nm. This group of samples is called the NaOH-treated samples.
The mass of the precipitate can be calculated by referring to the corresponding concentrations, and the experiment is conducted twice. The precipitate mass is acquired by subtracting the mass acquired from a non-NaOH sample from its corresponding NaOH-treated sample. The resulting number is multiplied by 2.143 to convert the mass of Si to SiO₂.

Results:

![](https://static.igem.wiki/teams/5685/newphoto/figure-18-1n.webp)

Figure 18.1. Blue color formation in NaOH-treated samples

![](https://static.igem.wiki/teams/5685/newphoto/figure-18-2n.webp)

Figure 18.2. Scanning samples under 825nm using a spectrophotometer

![](https://static.igem.wiki/teams/5685/silica/20250922235241.webp)

Figure 18.3. Precipitate samples after two rounds of centrifugation

Table 18.1. Data acquired across 4 non-NaOH samples

![](https://static.igem.wiki/teams/5685/last/2025-10-07-124823.webp)

Table 18.2. Data acquired across 4 NaOH-treated samples

![](https://static.igem.wiki/teams/5685/last/2025-10-07-124807.webp)

Table 18.3. Eventual mass of biosilica formed in different samples

![](https://static.igem.wiki/teams/5685/last/2025-10-07-124838.webp)

![](https://static.igem.wiki/teams/5685/silica/data-1.webp)

Figure 18.4. Mass of biosilcia formed in four different samples

Conclusion:
The ANOVA and t-tests reuslts suggest that the experimental results are not random and significant. By directly reacting TEOS with protein extracted using sonication, the results are more ideal than those acquired earlier. The tests are successful since the control groups show lower blue color formation and the mass of precipitate is also lower than that of the experimental groups, confirming the success of the plasmid design and the bacterial ability for silica formation. By calculating the mass difference between the non-NaOH and NaOH-treated groups, the impact of possible remaining TEOS is ruled out, since samples treated without NaOH only show the presence of TEOS but not silica.

**6. Sulfite reduction test for [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta**

Date: 19/09/2025

Aim:
To test whether [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta are capable of reducing sulfite.

Protocols:

Pipette 50uL bacteria solution to 1.5mL tube. Add 50uL cold 1M perchloric acid, mix thoroughly. Centrifuge at 1500g for 10 mins. Pipette 50uL supernatant fluid into a new 1.5mL tube. Add 25uL 1M NaOH into the new tube with supernatant fluid, mix thoroughly. Pipette 1.25mL distilled water, 250uL solution 1 (buffer), 50uL sample solution(prepared in step 1~5), 10uL solution 3 and 100uL solution 2 (NADH) into a 15mL tube, mix them well. Wait for 4 mins. Set spectrophotometer to 340nm, pipette 1mL solution into a curvette, record the OD. Pipette the solution back to the tube from the cuvette, use ddH2O, flush the cuvette for 3 times. Pipette 10uL suspension 4 (sulfite oxidase) and mix thoroughly after testing the OD, wait for 30 mins. Set spectrophotometer to 340nm, pipette 1mL solution into a cuvette, record the absorbance.

![](https://static.igem.wiki/teams/5685/sepnotebook/fig-18-1.webp)

Figure 18.5. Adding kit solution to samples.

Results:

Table 18.1. Data and sulfite concentration of each sample.

![](https://static.igem.wiki/teams/5685/sepnotebook/sulfite-table.webp)
![](https://static.igem.wiki/teams/5685/sepnotebook/sulfite-result.webp)

Figure 18.6. Sulfite concentration of each sample.

Conclusion:

The sulfite concentration of [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM, and [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta are lower than that of BL21-pET11a, but the deviation value of BL21-pET11a was high, indicating that our plasmids are capable of reducing sulfite.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block cover\_content %}{% endblock %}
{% block page\_content %}

## **Building a more accurate and reliable test for repairing - Silicon molybdenum Blue Colorimetric Method**

---

#### Please click the buttons below to show detailed information:

---

Aim & Design
Build and Learn (Cycle 1)
Build and Learn (Cycle 2)
Build and Learn (Cycle 3)
Build and Learn (Cycle 4)

#### **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:**

![](https://static.igem.wiki/teams/5685/silica2/4.webp)

Figure 3. OD values of four different samples

![](https://static.igem.wiki/teams/5685/silica2/data-2.webp)

Figure 4. Mass of biosilica formed in four different samples

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:**

![](https://static.igem.wiki/teams/5685/silica2/hhh.webp)

Figure 5. OD values of four different samples

![](https://static.igem.wiki/teams/5685/silica2/2.webp)

Figure 6. Mass of biosilica formed in four different samples

#### **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.

![](https://static.igem.wiki/teams/5685/silicaengineer/20251005122619.webp)

Figure 7. Visualization of the "mass subtraction concept".

**3.3.2 Tests/Results:**

![](https://static.igem.wiki/teams/5685/silica2/data-5.webp)

Figure 8. OD values of four different non NaOH samples

![](https://static.igem.wiki/teams/5685/silica2/data-9.webp)

Figure 9. OD values of four different NaOH treated samples

![](https://static.igem.wiki/teams/5685/silica2/data-3.webp)

Figure 10. Mass of biosilica formed in four different samples

#### **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:**

![](https://static.igem.wiki/teams/5685/silica2/data-8.webp)

Figure 11. OD values of four different non NaOH samples

![](https://static.igem.wiki/teams/5685/silica2/data-6.webp)

Figure 12. OD values of four different NaOH treated samples

![](https://static.igem.wiki/teams/5685/silica2/data-1.webp)

Figure 13. Mass of biosilica formed in four different samples

**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

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

## What Should this Page Contain?

---

Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block cover\_content %}{% endblock %}
{% block page\_content %}

#### Please click the buttons below to show detailed information:

---

Validation
Adhesion
Prevention
Repairing

## 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:

![](https://static.igem.wiki/teams/5685/protocol/data-1.webp)

Figure V1. qPCR result of [1]pET-J23119 INP-silicatein + INP-YFP-csgA

![](https://static.igem.wiki/teams/5685/protocol/data-2.webp)

Figure V2. qPCR result of [2]pET-J23119-INP-silicatein

![](https://static.igem.wiki/teams/5685/protocol/data-3.webp)

Figure V3. qPCR result of [3]pET-T7-INP-YFP-csgA

![](https://static.igem.wiki/teams/5685/protocol/data-4.webp)

Figure V4. qPCR result of [4]pET-J23119-ydeD (K4171005)

![](https://static.igem.wiki/teams/5685/protocol/data-5.webp)

Figure V5. qPCR result of [5]pET-J23119-yhaM

![](https://static.igem.wiki/teams/5685/protocol/data-6.webp)

Figure V6. qPCR result of [6]pET-T7-RBS-NSP4-sulfite reductase alpha -sulfite reductase beta

![](https://static.igem.wiki/teams/5685/protocol/data-7.webp)

Figure V7. qPCR result of [7]pET-T7-INP-YFP (K523013)-T7 tag

![](https://static.igem.wiki/teams/5685/protocol/data-8.webp)

Figure V8. qPCR result of [8]pET-T7-RBS-INP-silicatein (K1890001)-T7 tag

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:

![](https://static.igem.wiki/teams/5685/protocol/marked-igem-pc-2025-07-11-flag-1-2-4-5-composite.webp)

Figure V9. Successful Western blot result of [1]pET-J23119 INP-silicatein + INP-YFP-csgA, [2]pET-J23119-INP-silicatein and [5]pET-J23119-yhaM

![](https://static.igem.wiki/teams/5685/protocol/marked-igem-pc-2025-08-01-wb-3old-3new-composite-1.webp)

Figure V10. Successful Western blot result of [3]pET-T7-INP-YFP-csgA

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:

![](https://static.igem.wiki/teams/5685/julynotebook/flushing-test-result-v3.webp)

Figure A1. Bar graphs presenting colony counts (CFU) of BL21-pET11a ,[1] pET-J23119 INP-silicatein + INP-YFP-csgA, [3] pET-J23119-INP-YFP-csgA and [7]pET-T7-INP-YFP (K523013)-T7 tag in flushing tests under pH 3, 5, 7, and 9 conditions, with statistical significance determined by t-test.
(\*, 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).

![](https://static.igem.wiki/teams/5685/maynotebook/flushing-main.webp)

Figure A2. Bar graphs showing Flushing test result BL21-pET11a ,[1] pET-J23119 INP-silicatein + INP-YFP-csgA, [3] pET-J23119-INP-YFP-csgA and [7]pET-T7-INP-YFP (K523013)-T7 tag with different pH solutions and without flushing

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):**

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.

**[1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA:**

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.

**[3] pET-T7-INP-YFP(K523013)-csgA:**

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.

**[7] pET-T7-INP-YFP (K523013)-T7 tag:**

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.

![](https://static.igem.wiki/teams/5685/adhesion/bl21-pet11a-cfu.webp)

Figure A3. Fluorescence microscopy images of E. coli with BL21-pET11a (control) under conditions of without flushing and flushing with pH 3, 5, 7, 9 solutions.

![](https://static.igem.wiki/teams/5685/adhesion/1-cfu.webp)

Figure A4. Fluorescence microscopy images of E. coli with [1] pET-J23119 INP-silicatein (K1890001) + INP-YFP-csgA under conditions of without flushing and flushing with pH 3, 5, 7, 9 solutions.

![](https://static.igem.wiki/teams/5685/adhesion/3-cfu.webp)

Figure A5. Fluorescence microscopy images of E. coli with [3] pET-T7-INP-YFP(K523013)-csgA under conditions of without flushing and flushing with pH 3, 5, 7, 9 solutions.

![](https://static.igem.wiki/teams/5685/adhesion/7-cfu.webp)

Figure A6. Fluorescence microscopy images of E. coli with [7] pET-T7-INP-YFP (K523013)-T7 tag under conditions of without flushing and flushing with pH 3, 5, 7, 9 solutions.

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:

![](https://static.igem.wiki/teams/5685/resultprevention/sulfate-reduction-pathway-updated.webp)

Figure P1. Sulfate reduction pathway in bacteria [2]

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.

![](https://static.igem.wiki/teams/5685/resultprevention/sulfate-reduction-pathway-with-enzymes.webp)

Figure P2. Sulfate reduction pathway in bacteria with cysteine desulfhydrase enzymes' name [3]

#### 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:

![](https://static.igem.wiki/teams/5685/protocol/20251003190518-2243-54.webp)

Figure P3. Result of qPCR of cysteine desulfhydrase enzymes

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:

![](https://static.igem.wiki/teams/5685/augustlogbook/p2.webp)

Figure P4. BL21-pET11a growth with different concentrations of sodium sulfate added

![](https://static.igem.wiki/teams/5685/resultprevention/sulfite-growth-curve.webp)

Figure P5. BL21-pET11a growth with different concentrations of sodium sulfite added

![](https://static.igem.wiki/teams/5685/augustlogbook/p3.webp)

Figure P6. BL21-pET11a growth with different concentrations of sulfuric acid added

#### Conclusion:

This pre-experiments test indicates the concentrations of sulfate and sulfite in the tested range do not have a significant effect on bacterial growth

### **Sulfate 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.).
![](https://static.igem.wiki/teams/5685/augustlogbook/p4.webp)

Figure P7. Chemical equation of reaction 1

Hydrogen peroxide formed in this reaction is reduced in the presence of the enzyme, NADH peroxidase and NADH (Shown in Figure P8.).

![](https://static.igem.wiki/teams/5685/augustlogbook/p5.webp)

Figure P8. Chemical equation of reaction 2

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:

![](https://static.igem.wiki/teams/5685/resultprevention/sulfite-reduction-test-result.webp)

Figure P9. Sulfite concentration after adding 1mM sodium sulfite into bacterial culture for 48 hours

![](https://static.igem.wiki/teams/5685/resultprevention/sulfate-reduction-test.webp)

Figure P10. Sulfate concentration after adding 1mM sulfuric acid into bacterial culture for 48 hours

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](https://store.sangon.com/productDetail?productInfo.code=D799571) 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:

![](https://static.igem.wiki/teams/5685/resultprevention/cysteine-control.webp)

Figure P11. Cysteine amount of samples without adding extra cysteine

![](https://static.igem.wiki/teams/5685/resultprevention/2mm-cysteine.webp)

Figure P12. Cysteine amount of samples with 2mM cysteine added

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](https://m.boxbio.cn/product/838.html?goodsno=AKSM001C) 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:

![](https://static.igem.wiki/teams/5685/prevention/2025-10-06-004625.webp)

Figure P13. Amount of Hydrogen sulfide of YhaM and BL21-pET11a (control group)

(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**

* **[8] pET-T7-RBS-INP-silicatein (K1890001)-T7 tag**

*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:

![](https://static.igem.wiki/teams/5685/silica/2.webp)

Figure R1. The relationship between the silicon concentrations of the samples and their OD values at 825 nm

#### 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.

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