March to May

All students were chosen as participants for iGEM 2024.

April

During our initial training on synthetic biology and iGEM, guided by our principal investigator, we explored the evolution of synthetic biology, its core definitions, the basics of entrepreneurship and AI, and insights into the iGEM competition. Furthermore, we held a brainstorming session to generate ideas for our project during this phase.

May

We engaged in specialized courses covering molecular biology, genetic engineering, biochemistry, biological research techniques, and mathematical modeling. We also covered laboratory safety and the theoretical foundations of our experiments, finalized our plasmid designs, and submitted requests for DNA synthesis to a provider. With this knowledge, we began the comprehensive design of our project in May, focusing on aspects like experimental setup and plasmid construction. Additionally, we organized protocols and gathered the necessary materials for our summer experiments.

We recorded each day spent conducting experiments in the lab. Since the experimental schedule did not consist of continuous days, we did not use a standard diary format. "Lab day x" refers to the x-th day of work in the lab. The experimental period runs from June 3, 2024, to August 13, 2024.

We designed the plasmids for the project and sent them to Sangon Biotech for DNA synthesis and vector construction, with a synthesis period of two weeks. Upon receiving the plasmids, we performed double enzyme digestion verification, conducted transformation, and picked single colonies for sequencing verification. Lab Day began after we completed the plasmid construction, with the main task being testing.

Lab Day 1:

Stock Solution Preparation

1. 50 mg/ml Kanamycin(Kana) Stock Reagent
2. 50 mg/ml Streptomycin (Ste) Stock Reagent
3. 100 mM IPTG Stock Reagent
4. 10 mg/ml Congo Red (CR) Stock Reagent
5. 10 mg/ml Coomassie Brilliant Blue Stock Reagent
6. LB Medium Preparation
7. LB 平板配置
8. Congo Red Plate Preparation

Group	10 mg/ml Congo Red Stock	10 mg/ml Coomassie Brilliant Blue Stock	50 mg/ml Kanamycin Stock	100 mM IPTG Stock
Group 1	2.5 ml	50 µl	1 ml	0 ml
Group 2	2.5 ml	50 µl	1 ml	0.5 ml
Group 3	2.5 ml	50 µl	1 ml	2.5 ml
Group 4	2.5 ml	50 µl	1 ml	4 ml

Bacterial Resuscitation:

1. Take the glycerol stock of Curlis fiber - pET28a+ and PET28a+ vector from the -80°C freezer, streak onto a kanamycin-resistant plate, and incubate overnight at 37°C.
2. Take the glycerol stock of sfGFP-SpyCatcher and amliCP-SpyCatcher from the -80°C freezer, streak onto a streptomycin-resistant plate, and incubate overnight at 37°C.

Lab Day 2:

1. Pick 3 single colonies from the Curlis fiber - pET28a+ and pET28a+ plates into 5 ml of LB with kanamycin, and incubate overnight at 220 rpm and 37°C.
2. Pick 3 single colonies from the sfGFP-SpyCatcher and amliCP-SpyCatcher plates into 5 ml of LB with streptomycin, and incubate overnight at 220 rpm and 37°C.

Lab Day 3:

Extract plasmids from the overnight cultures, measure the concentration, and send the remaining plasmid solution to Ansenda for Sanger sequencing.

Lab Day 4:

Sequencing Verification

Bacterial Transformation:

1. Transform Curlis fiber - pET28a+ and pET28a+ into BL21(DE3) cells, plate on kanamycin plates, and incubate overnight at 37°C.
2. Transform sfGFP-SpyCatcher and amliCP-SpyCatcher into BL21(DE3) cells, plate on streptomycin plates, and incubate overnight at 37°C.

Lab Day 5:

1. Pick 3 single colonies from the Curlis fiber - pET28a+ plate into 3 ml of LB with kanamycin, and incubate overnight at 220 rpm.
2. Forgot to pick a single colony from the pET28a+ empty vector plate
3. Pick 1 single colony from the sfGFP-SpyCatcher and amliCP-SpyCatcher plates into 50 ml of LB with streptomycin, and incubate overnight at 220 rpm (expression of GFP (green) and amliCP (blue) was observed on the plates).

Lab Day 6:

Congo Red Experiment:

1. Take Congo red plates with different concentrations of IPTG, drop 50 µl onto the plates, and incubate at room temperature.

Fluorescent Protein Experiment:

1. Take the overnight cultures of sfGFP-SpyCatcher and amliCP-SpyCatcher from the shaker, and collect 100 µl as a sample for whole cell analysis.
2. Transfer to a 50 ml conical flask and centrifuge at 10,000 rpm for 5 minutes.
3. Add 8 ml of Beyotime non-denaturing lysis buffer, resuspend the pellet thoroughly, and place it on an ice bath. Sonicate for 15 minutes to lyse the cells.
4. Centrifuge at 10,000 rpm for 5 minutes, and observe that a large amount of bacteria remains unlysed.
5. Replace with denaturing lysis buffer, resuspend the bacteria, and place on an ice bath. Sonicate for 15 minutes, resulting in a large amount of foam.
6. Centrifuge at 10,000 rpm for 5 minutes and collect the supernatant.
7. Take 100 µl of the supernatant as a sample, and store all samples at 4°C, awaiting column purification.

Lab Day 7:

Congo Red Plate Preparation

1. Eight bottles of BeyoPure™ LB Broth with Agar (Catalog No. ST158) were opened and added to eight 1L blue-capped bottles. Ultrapure water from the Milli-Q® Type 1 Ultrapure Water System was added to a final volume of 500 ml per bottle. The solutions were shaken well, autoclave tape was applied, the bottle caps were loosened, and the solutions were sterilized at 121°C for 30 minutes. After cooling to approximately 60°C, the corresponding antibiotic, Congo Red, Coomassie Brilliant Blue, and IPTG stock solutions were added as follows:

Group	10 mg/ml Congo Red Stock	10 mg/ml Coomassie Brilliant Blue Stock	50 mg/ml Kanamycin Stock	100 mM IPTG Stock
Group 1	2.5 ml	50 µl	1 ml	0 ml
Group 2	2.5 ml	50 µl	1 ml	0.5 ml
Group 3	2.5 ml	50 µl	1 ml	2.5 ml
Group 4	2.5 ml	50 µl	1 ml	5 ml
Group 5	0 ml	0 ml	1 ml	0 ml
Group 6	0 ml	0 ml	1 ml	0.5 ml
Group 7	0 ml	0 ml	1 ml	2.5 ml
Group 8	0 ml	0 ml	1 ml	5 ml

Protein Purification:

1. The BeyoGold™ His-tag Purification Resin (denaturant-resistant) was loaded into the column, and the purification column was equilibrated twice with 1 ml of lysis buffer.
2. The supernatant was loaded onto the column. To maximize yield, the flow-through was collected and reloaded onto the column 3-5 times to ensure complete binding of the His-tagged protein.
3. The column was washed five times with 1 ml of denaturing lysis buffer.
4. The column was then washed five times with 1 ml of washing buffer.
5. The bound protein was eluted 6-10 times with 0.5 ml of elution buffer, and the final eluate was collected.

Lab Day 8:

Congo Red Experiment:

1. Take 30 µl from the overnight cultures of Curlis fiber - pET28a+ and pET28+, and gently transfer them onto Congo red plates and normal plates containing different concentrations of IPTG.
2. Let the plates sit for 1 hour until the LB liquid has completely dried. Then, invert the plates and incubate them at 30°C for 48 hours.

SDS-PAGE:

1. Prepare the samples: take the 5X loading buffer from the -20°C freezer and thaw it. Set the water bath to 99°C.
2. Take 50 ml of 20X MOPS buffer and add it to 950 ml of ddH2O to prepare 1L of 1X MOPS buffer.
3. Take 40 µl of each sample stored in the 4°C fridge:
1. GFP whole cell
2. GFP supernatant before purification
3. GFP purified protein
4. Amlicp whole cell
5. Amlicp supernatant before purification
6. Amlicp purified protein
4. Add 10 µl of loading buffer, vortex to mix thoroughly, and boil the samples in a 99°C water bath for 10 minutes.
5. Centrifuge at 12,000 rpm for 3 minutes.
6. Take the pre-cast Young gel from the 4°C fridge, remove the insulating tape, place it in the Bio-Rad electrophoresis chamber, and remove the comb.
7. Load 10 µl of each sample into the wells, following the order: whole cell, supernatant before purification, and supernatant after purification.
8. A leak occurred.
9. After replacement, the leakage still occurred. We contacted the manufacturer, and they agreed to provide a replacement. The experiment was paused, and the samples were stored in a 4°C refrigerator for future use.

Lab Day 9:

Congo Red Experiment:

1. Take photos.
2. Observations:
1. The bacterial colonies on the regular LB agar plates are all white.
2. On the Congo red plates with IPTG, the colonies carrying the Curlis Fiber plasmid are generally redder than those without the Curlis Fiber plasmid, indicating that Curlis Fiber is being expressed. This suggests that we can proceed with the liquid experiment.

Curlis Fiber Liquid Expression Experiment:

1. Pick three single colonies from each of the Curlis Fiber-pET28a+ and pET28a+ vector plates into 3 ml of LB with kanamycin, and incubate overnight at 220 rpm and 37°C.

Lab Day 10:

Curlis Fiber Liquid Expression Experiment:

1. Take 40 µl from each of the Curlis Fiber-pET28a+ and pET28a+ vector overnight cultures and add it to 4 ml of LB with kanamycin. For each group, repeat 3 times to obtain a total of 9 tubes of bacterial culture. Incubate at 220 rpm and 37°C for 2 hours.
2. After 2 hours, add 0 µl, 4 µl, and 40 µl of 100 mM IPTG to the cultures in the logarithmic phase to achieve final IPTG concentrations of 0 mM, 0.1 mM, and 1 mM, respectively. The specific grouping details are as follows:

Plasmid Group	IPTG Concentration	IPTG Concentration	IPTG Concentration
Curlis Fiber-pET28a+ -1	0mM	0.1 mM	1mM
Curlis Fiber-pET28a+ -2	0mM	0.1 mM	1mM
Curlis Fiber-pET28a+ -3	0mM	0.1 mM	1mM
pET28a+ vector-1	0mM	0.1 mM	1mM
pET28a+ vector-2	0mM	0.1 mM	1mM
pET28a+ vector-3	0mM	0.1 mM	1mM

Place in a 30°C shaker and incubate at 220 rpm for 24 hours.

Lab Day 11:

SDS-PAGE (POI):

1. Prepare the samples: take the 5X loading buffer from the -20°C freezer and thaw it. Set the water bath to 99°C.
2. Take 50 ml of 20X MOPS buffer and add it to 950 ml of ddH2O to make 1L of 1X MOPS buffer.
3. Take 40 µl of each sample from the 4°C refrigerator:
1. GFP whole cell
2. GFP supernatant before purification
3. GFP purified protein
4. Amlicp whole cell
5. Amlicp supernatant before purification
6. Amlicp purified protein
4. Add 10 µl of loading buffer, vortex to mix thoroughly, and boil in the 99°C water bath for 10 minutes.
5. Centrifuge at 12,000 rpm for 3 minutes.
6. Take the Young pre-cast gel from the 4°C refrigerator, remove the insulating tape, place it in the Bio-Rad electrophoresis chamber, and remove the comb.
7. Load 10 µl of each sample into the wells in the order of whole cell, supernatant before purification, and supernatant after purification.
8. Finally, add 5 µl of the protein marker.
9. Run the electrophoresis at 200 mV for 20 minutes.
10. Disassemble the gel plates and add 50 ml of Coomassie Brilliant Blue rapid stain solution. Stain at 80 rpm for 30 minutes.
11. Pour off the Coomassie Brilliant Blue solution, wash several times with ddH2O, then add sufficient ddH2O and shake at room temperature at 80 rpm overnight.

Lab Day 12:

Curlis Fiber Liquid Expression Experiment:

1. Take photos.
2. It can be observed that filamentous precipitates appeared in both the 0.1 mM and 1 mM groups of Curlis fiber - pET28a, while no such precipitates were seen in the pET28a vector and Curlis fiber - pET28a 0 mM groups. This suggests that the filamentous precipitates are likely the produced CurlisFiber.
3. Using a pipette tip, extract the filamentous precipitate from the bacterial culture and transfer it to 1 mL of ddH2O and 1 mL of 1X PBS. After pipetting to mix, the filamentous material remains insoluble.
4. Eventually dissolves.
5. Store the remaining bacterial cultures in the 4°C refrigerator.

Lab Day 13:

SDS-PAGE（Curlis）:

1. Prepare the samples: take the 5X loading buffer from the -20°C freezer and thaw it. Set the water bath to 99°C.
2. Take 50 ml of 20X MOPS buffer and add it to 950 ml of ddH2O to make 1L of 1X MOPS buffer.
3. Take 40 µl of each sample from the 4°C refrigerator:
1. Bacterial culture
2. Curlis fiber guanidine hydrochloride solution.
4. Add 10 µl of loading buffer, vortex to mix thoroughly, and boil in a 99°C water bath for 10 minutes.
5. Centrifuge at 12,000 rpm for 3 minutes.
6. Take the Young pre-cast gel from the 4°C refrigerator, remove the insulating tape, place it in the Bio-Rad electrophoresis chamber, and remove the comb.
7. Load 10 µl of each sample into the wells.
8. Finally, add 5 µl of protein marker.
9. Run the electrophoresis at 200 mV for 20 minutes.
10. Disassemble the gel plates and add 50 ml of Coomassie Brilliant Blue rapid stain solution. Stain at 80 rpm for 30 minutes.
11. Pour off the Coomassie Brilliant Blue solution, wash several times with ddH2O, then add sufficient ddH2O and shake at room temperature at 80 rpm overnight.

Rerun the Curlis fiber samples, but the wells are still blocked.

It is suspected that the 8M guanidine hydrochloride is causing the blockage, as it tends to crystallize and precipitate at room temperature.

Dilute the 8M guanidine hydrochloride solution with ddH2O in a gradient to obtain 80 µl of 8M, 4M, and 2M guanidine hydrochloride solutions. Add 20 µl of loading buffer to each, boil at 99°C for 10 minutes, and centrifuge at 10,000 rpm for 3 minutes.

It was observed that needle-like precipitates appeared in all the solutions, indicating that 8M guanidine hydrochloride cannot be used for loading. The plan is to perform a column purification step on the Curlis fiber solution before proceeding with further analysis.

Lab Day 14:

Column Purification of Curlis Fiber Guanidine Hydrochloride Solution:

1. The BeyoGold™ GST-tag Purification Resin (denaturant-resistant) was loaded into the column, and the purification column was equilibrated twice with 1 ml of lysis buffer.
2. The supernatant was loaded onto the column. To maximize yield, the flow-through was collected and reloaded onto the column 3-5 times to ensure complete binding of the His-tagged protein.
3. The column was washed five times with 1 ml of denaturing lysis buffer.
4. The column was then washed five times with 1 ml of washing buffer.
5. The bound protein was eluted 6-10 times with 0.5 ml of elution buffer, and the final eluate was collected.

Lab Day 15:

Western Blot

1. Sample Preparation:
1. Collected the purified Curlis Fiber protein from the elution fractions after column purification.
2. Added 20 µl of protein sample to 5 µl of 5X loading buffer.
3. Heated the samples at 99°C for 10 minutes to denature the proteins.
4. Centrifuged at 12,000 rpm for 3 minutes to remove any debris.
2. SDS-PAGE:
1. Prepared a 12% SDS-PAGE gel.
2. Loaded 10 µl of each protein sample into individual wells.
3. Loaded 5 µl of the protein marker into one well.
4. Ran the gel at 120V for 1 hour until the dye front reached the bottom of the gel.
3. Protein Transfer:
1. Soaked the SDS-PAGE gel and nitrocellulose membrane in 1X transfer buffer.
2. Assembled the transfer sandwich (sponge, filter paper, gel, nitrocellulose membrane, filter paper, sponge).
3. Transferred proteins at 100V for 1.5 hours at 4°C.
4. Blocking:
1. After transfer, incubated the nitrocellulose membrane in 5% BSA blocking buffer for 1 hour at room temperature with gentle shaking to block non-specific binding sites.
5. Primary Antibody Incubation:
1. Incubated the membrane overnight at 4°C with anti-His tag primary antibody diluted 1:1000 in TBST.
6. Secondary Antibody Incubation:
1. Washed the membrane three times with TBST (10 minutes each).
2. Incubated the membrane with HRP-conjugated secondary antibody (diluted 1:5000 in TBST) for 1 hour at room temperature.
7. Washing:
1. Washed the membrane again three times with TBST (10 minutes each).
8. Detection:
1. Applied ECL detection reagent to the membrane and incubated for 1 minute.
2. Visualized the protein bands using the Bio-Rad ChemiDoc imaging system.

Results:

- No visible bands were observed for the Curlis Fiber protein, even after several exposure attempts.

Lab Day 16:

Western blot successful.

Lab Day 17:

Pick single colonies for expanded culture to express Curlis.

Lab Day 18:

Induced expression.

Lab Day 19:

Collect and purify Curlis.

Lab Day 20:

Centrifuge at 12,000 rpm, 4°C for 10 minutes, transfer the supernatant to a new 1.5 ml centrifuge tube, and keep on ice. Then, use the BCA method to determine the protein concentration.

Take a small amount of the supernatant, denature it, and use it for the input experiment, i.e., WB (Western Blot) to detect the target protein.

Lab Day 21:

Add 1.0 μg of IgG (normal IgG from the same species as the antibody used for precipitation in the IP experiment) and 20 μL of Protein A/G beads (fully mixed before use) to the protein supernatant of the negative control (IgG) group.

For the experimental group, directly add 20 μL of Protein A/G beads. Incubate on a rotator at 4°C for 1 hour.

Centrifuge at 2000g, 4°C, for 5 minutes, and collect the supernatant.

Add 1-10 μL (0.2-2 μg) of the antibody, and incubate at 4°C overnight.

Lab Day 22:

Add 80 μL of Protein A/G beads (fully mixed before use), gently flick to mix, and incubate at 4°C for 2 hours.

Centrifuge at 2000g, 4°C, for 5 minutes, carefully remove the supernatant, being cautious not to disturb the beads at the bottom, and collect the immunoprecipitate complex.

Wash the immunoprecipitate complex 4 times with 1 ml of pre-chilled IP lysis buffer (without adding inhibitors), centrifuging at 2000g, 4°C, for 5 minutes after each wash, carefully discarding the supernatant each time.

After the final wash, carefully remove as much supernatant as possible, then add 80 μL of 1× reducing loading buffer, boil for 10 minutes, and centrifuge at 1000g, 4°C, for 5 minutes to collect the supernatant.

Lab Day 23:

IB Detection

Take 10 µL of the supernatant sample for WB (Western Blot) analysis.

For detailed analysis of the results, refer to the Results section.

August & September

Data analysis, wiki documentation, scientific illustration, and data fitting have been completed.

October

We submitted the wiki and presentation video, and we will participate in the Jamboree in person.