Overview
During our iGEM year, we organized our project by performing wet lab experiments.
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.
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
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).
Conclusion:
plasmids Midi of [2]-[6] & [8] were successful, verifying that the bacterial clones contained the correct plasmids with our desired inserts.
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.
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.
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.
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
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
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.
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.
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:
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.
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.
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.
Results:
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.
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
Results:
The cysteine destruction test failed.
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.
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.
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
[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.
The bacterial growth test failed.
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.
Successful growth of bacteria BL21-pET11a under different concentrations of sulfate.
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.
Results:
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.
Results:
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.
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.
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.
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.
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:
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.
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:
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.
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.)
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.
Result:
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.
Result:
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.
Result:
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:
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:
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
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.
Result:
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.
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
Result:
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
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.
Result:
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
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.
Results:
Conclusion:
The ANOVA and t-test results indicate that the experimental results are relatively random and insignificant. The test fails because the control group shows a larger amount of precipitate, indicating more silica formation, which is abnormal since it does not express silicatein. According to Figure 16.1, the ANOVA test and all t-tests are non-significant, indicating that the absorbance readings for all silicatein constructs are not statistically different from the control. In the mass quantification of biosilica (Figure 16.4.), the ANOVA test and the individual t-tests show that none of the engineered samples produce a mass of biosilica that is statistically different from the control. The experiment does not confirm the functional viability of our plasmids for silicatein biomineralization.
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: 01/09/2025
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. 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: 02-03/09/2025
Aim:
Prepare for the sulfite and sulfate reduction test.
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.
4. 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: 04-05/09/2025
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. On 5th September, we used the microplate assay method, where 250uL solution 1 (buffer), 100uL solution 2(NADH), 5uL sample solution, 125uL distilled water, 10uL solution 3 and 10uL solution 4 were added to a well in the 96 well flat-bottom plate, and spectrophotometric multiwell plate reader was used to record OD.
Results:
The deviation values for BL21-pET11a, [4]pET-J23119-ydeD (K4171005), [5]pET-J23119-yhaM groups for the first time using 96 well-plates were relatively high, therefore we decided to conduct the experiment again. Moreover, we decided to prepare a sample on ice to lower the speed of sulfite oxidizing into sulfite oxidase.
5. 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: 04-05/09/2025
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.
Protocol:
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 200u , 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, 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.
Result:
In the first test, the concentration of [4]ydeD broth was significantly lower than that of the other groups, matching with the qPCR result of the sulfate reduction pathway enzymes. Moreover, the concentration of [5]yhaM and [6]sulfite reductase broth were also lower than the BL21-pET11a control group. However, in the second test, the concentrations of sulfate for each group were similar. In the third test, the concentration of sulfate in [6]sulfite reductase broth was obviously lower.
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.
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:
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.
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:
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.
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.