iGEM 2024 — Team lcg-china

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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# Notebook

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![Cover Image](https://static.igem.wiki/teams/5461/page-first-img/notebook2.jpg)

Notebook

##

LCG-China Team's iGEM 2024 Journey: From Formation to Jamboree

We embarked on our iGEM 2024 journey by intensively recruiting team members and undergoing rigorous initial training through May, all under the expert guidance of our coaching team. During this phase, we delved into the essentials of synthetic biology, which set the stage for us to peer review iGEM projects and develop a modular, decoupled system that effectively binds proteins of interest to bacterial cellulose—tackling significant compatibility challenges. Throughout the summer, we thoroughly tested this system in the lab, advancing from plasmid design to extensive experimental validation. Additionally, we explored the system's entrepreneurial applications and investigated AI safety issues in synthetic biology. Our journey will culminate in October at the iGEM Jamboree, where we aim to showcase our innovative project and its potential global impact.

Check our timeline on this notebook.

![](https://static.igem.wiki/teams/5461/icon/fen.png)Before Summer
![](https://static.igem.wiki/teams/5461/icon/cheng.png)March to May
![](https://static.igem.wiki/teams/5461/icon/cheng.png)April
![](https://static.igem.wiki/teams/5461/icon/cheng.png)May
![](https://static.igem.wiki/teams/5461/icon/fen.png)Summer Stage
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 1:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 2:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 3:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 4:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 5:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 6:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 7:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 8:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 9:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 10:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 11:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 12:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 13:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 14:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 15:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 16:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 17:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 18:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 19:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 20:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 21:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 22:
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Lab Day 23:
![](https://static.igem.wiki/teams/5461/icon/fen.png)After Summer
![](https://static.igem.wiki/teams/5461/icon/cheng.png)August & September
![](https://static.igem.wiki/teams/5461/icon/cheng.png)October

![](https://static.igem.wiki/teams/5461/icon/zi.png)Back to the top

## Before Summer

#### 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.

## Summer Stage

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.

![](https://static.igem.wiki/teams/5461/freeze/experiments-prepare.png)

A glimpse of our wet-lab experiments

#### 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.

## After Summer

#### 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.

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block cover %}

# Engineering Success

{% endblock %}
{% block page\_content %}

![Cover Image](https://static.igem.wiki/teams/5461/page-first-img/engineering-big.jpg)

Engineering

##

Modular System for Efficient Bacterial Cellulose Composite Modification through Multiple DBTL Cycles

**Bacterial cellulose**, as a natural polymer material, holds great potential across various fields due to its abundance, renewability, and biocompatibility[1]. This year, we developed a modular, decoupled approach to bacterial cellulose composite modification, designed to streamline and improve the efficiency of protein-based modifications. The system is comprised of three core modules (illustrated at the top of this page). Each module was rigorously design, build, tested and iterated through multiple DBTL (Design-Build-Test-Learn) cycles, and we successfully validated all three modules, both individually and as a combined system. This comprehensive process led to the achievement of our **Engineering Success**. Additionally, we discussed future directions for expanding this work, providing a foundation upon which future iGEM teams can build.

![](https://static.igem.wiki/teams/5461/icon/fen.png)Engineering Architecture
![](https://static.igem.wiki/teams/5461/icon/fen.png)Scaffold Protein
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Cycle 1
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Cycle 2
![](https://static.igem.wiki/teams/5461/icon/fen.png)Target Fusion Protein
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Cycle 1
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Cycle 2
![](https://static.igem.wiki/teams/5461/icon/fen.png)Optimization of BC Production
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Cycle 1
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Cycle 2
![](https://static.igem.wiki/teams/5461/icon/fen.png)Compostion
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Cycle 1
![](https://static.igem.wiki/teams/5461/icon/fen.png)Work Going on
![](https://static.igem.wiki/teams/5461/icon/fen.png)Referrence

![](https://static.igem.wiki/teams/5461/icon/zi.png)Back to the top

## Engineering Architecture

The following illustration shows the architecture of our Bacterial Cellulose Modification System:

![](https://static.igem.wiki/teams/5461/engineering/architecture.jpg)

**Module I. Bacterial Cellulose Production**

*Komagataeibacter* is a microorganism widely utilized in large-scale bacterial cellulose production[2]. We have optimized the production process, particularly by refining nitrogen source selection and cultivation conditions, thereby significantly improving both the yield and quality of the produced bacterial cellulose.

**Module II. Scaffold Protein Design and Production**

Through genetic engineering, we successfully constructed an *Escherichia coli* strain that expresses a scaffold protein, ***Curlis Fiber-Spytag***, capable of binding to bacterial cellulose. In this system, the *Curlis Fiber* domain provides the bacterial cellulose-binding functionality, while *Spytag* enables the heterologous linkage via the SpyCatcher system.

**Module III. Target Protein Design and Production**

We designed a ***SpyCatcher*-POI (protein of interest) fusion** with a standardized interface for expression and purification. Using two distinct color proteins as demonstration models, we validated both the expression and purification processes. In future applications, we anticipate that this approach will allow the facile substitution of various POIs through the Golden Gate cloning method.

**Composition**

Finally, our experimental results confirmed the high-efficiency binding of bacterial cellulose, the scaffold protein *Curlis Fiber-Spytag*, and the target protein *SpyCatcher*-POI. These findings demonstrate the effectiveness and feasibility of this modular system, providing a POC and novel strategy for the modification and application of bacterial cellulose.

**Work going on**

Future teams can focus on identifying more protein pairs with strong binding affinity, like SpyTag-SpyCatcher, and adopt iGEM 2022 Michigan’s molecular chaperone strategy to improve the expression of challenging proteins, such as antimicrobial peptides, in *E. coli*.

Check the detailed DBTL of each module and the composition, in the following contents.

## Expression Test and Purification of Scaffold Protein (Module II)

#### Cycle 1: Expression Test of *Curlis-Spytag*

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-d-n.png)

Through reviewing previous iGEM projects[3-6] and related literature[7], we discovered that Curli fibers, composed of the CsgA protein, have excellent protein fiber production characteristics and a strong binding ability to cellulose. Therefore, this year we linked the CsgA protein with the SpyCatcher peptide through a linker, constructing a fusion protein to be used as a scaffold protein in cellulose modification. This fusion protein was cloned into the pET28a+ vector for expression.

Since the CsgA protein, which forms Curli fibers, exists in an insoluble state after self-assembly and cannot be verified by conventional electrophoresis, we used Congo red staining as a preliminary validation method to detect its expression in Escherichia coli.

![](https://static.igem.wiki/teams/5461/engineering/figure-1.png)

*Figure1. Plasmid structure of Curlis Expression Module*

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-b-n.png)

1. **Plasmid Synthesis and Transformation:** The plasmid was obtained through full synthesis and transformed into *Escherichia coli* BL21(DE3). Three single colonies were picked and cultured overnight in LB medium containing antibiotics. Part of the bacterial solution was sent to Anshengda for sequencing confirmation, while the remaining solution was stored in 30% glycerol for future experiments.
2. **Congo Red Staining Plate Preparation:** Congo red plates with different IPTG concentrations were prepared to detect the expression and self-assembly of Curli fibers. Detailed experimental steps and concentration gradients can be found in the "Experiment" section.
3. **Control Experiment:** LB agar plates with different IPTG concentrations were also prepared as a control group to compare expression levels under different IPTG induction conditions. Detailed experimental steps and concentration gradients can be found in the "Experiment" section.

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-t-n.png)

1. **Bacterial Culture:** Three single colonies of Curlis-Spytag were picked from the plate and cultured overnight in LB medium with kanamycin. At the same time, a strain containing the empty pET28a+ plasmid was picked as a control group and cultured overnight in LB-kanamycin medium.
2. **Bacterial Solution Dropping and Incubation:** 30 µL of the overnight culture was dropped onto Congo red plates with different IPTG concentrations and onto the control plates. The plates were incubated at 30°C for 48 hours to observe colony color changes and self-assembly.

By observing the results of Congo red staining, we can preliminarily validate the expression of the CsgA-SpyCatcher fusion protein in *Escherichia coli* and the formation of Curlis fibers, providing data support for subsequent experiments.

![](https://static.igem.wiki/teams/5461/engineering/figure-2.png)

*Figure 2. Congo Red Staining Experiment Detecting the Expression and Self-Assembly of Curlis-Spytag Protein
In this experiment, bacterial strains expressing Curlis fiber, along with strains carrying the empty pET28a+ plasmid, were grown on Congo red plates (top image) and control plates (bottom image) with varying concentrations of IPTG: 0mM, 0.5mM, and 1mM. The differences in growth patterns and color changes on these plates reveal the expression and self-assembly of Curlis-Spytag. The Congo red staining serves as an indicator, highlighting the structural assembly of Curli fibers, offering a visual comparison between the experimental and control groups under different induction conditions.*Nata de coco milk tea [9]

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-l-n.png)

Through a simple Congo red staining experiment combined with extensive control experiments, we successfully validated that the designed plasmid could induce Escherichia coli to normally express Curlis fibers under the induction conditions. The experimental results showed that the induction effect was optimal at an IPTG concentration of 0.5 mM, which may represent the best induction concentration. Future work will focus on further optimizing Curlis fiber expression in liquid culture environments to collect and purify Curlis fibers, preparing for subsequent binding experiments.

#### Cycle2: Expression of Curlis-Spytag

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/2-d.png)

After completing the identification of solid-phase expression, we now turn to liquid culture, as it allows for more efficient collection of the target protein. The plasmid design remains the same. To validate our approach, we first designed a small-scale 4 mL liquid culture system to optimize the expression of Curlis fibers, facilitating more efficient collection and subsequent purification.

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/2-b.png)

Samples were taken from the previously sequenced and verified Curlis-Spytag glycerol stock and streaked onto LB plates containing kanamycin to isolate single colonies. At the same time, a strain carrying the empty pET28a+ plasmid was used as a control and similarly streaked onto LB plates containing kanamycin for single colony isolation.

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/2-t.png)

Three single colonies of Curlis-Spytag were picked from the plate and inoculated into LB medium containing kanamycin. The cultures were grown overnight at 37°C and 220 rpm. The next day, the overnight cultures were diluted 1:100 and grown until the OD600 reached 0.3, at which point different concentrations of IPTG were added for induction. The cultures were then incubated at 30°C and 220 rpm for another 24 hours.

After 24 hours, we observed the appearance of obvious filamentous precipitates in the bacterial cultures. This indicates that Curlis fibers were successfully expressed and self-assembled into fibrous structures in the liquid culture system, providing a solid basis for subsequent protein collection and functional validation.

![](https://static.igem.wiki/teams/5461/engineering/figure-3.png)

*Figure 3. Self-assembly of Curlis Fibers in Liquid Culture Medium
a. Growth status of bacterial strains expressing Curlis fibers and the filamentous precipitates (Curlis fibers) in liquid culture medium with 0mM, 0.1mM, and 1mM IPTG concentrations.
b. Growth status of the strain carrying the empty pET28a+ plasmid, and two single colonies expressing Curlis fibers, as well as the filamentous precipitates (Curlis fibers), in liquid culture medium with 0mM, 0.1mM, and 1mM IPTG concentrations.*

After washing with ddH2O, 1X PBS, and 5X PBS, we found that the filamentous precipitates in the culture medium could not be dissolved. To dissolve the Curlis fibers for detection using electrophoresis, we referred to literature and decided to use 8M guanidine hydrochloride, which successfully dissolved the precipitates. Subsequently, we purified the dissolved protein solution using a GST purification kit from Beyotime to remove non-specific proteins and obtain high-purity Curlis target protein.

Next, we performed Western blot (WB) analysis on the purified Curlis protein samples. Our designed Curlis-Spytag fusion protein carries a GST tag, and the target protein bands were detected using an anti-GST antibody. The results are shown in the following figure:

![](https://static.igem.wiki/teams/5461/engineering/figure-4.png)

*Figure 4. Identification of the Expression of Curlis Protein with Fusion Tag
Sample 1: Curlis-Spytag single colony strain No.1, Control: Negative control group.*

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/2-l.png)

1. The plasmid we designed can successfully express Curlis-Spytag in a liquid culture system, and the protein can be secreted extracellularly and self-assemble into fibrous structures.
2. The Curlis-Spytag fibers can be effectively dissolved into a soluble state using 8M guanidine hydrochloride, facilitating subsequent experiments such as protein purification, validation, and cellulose binding.

## Expression Test and Purification of Target Fusion Protein (Module III)

#### Cycle 1: Construction of the Modified Protein Expression System and Demonstration of Expression and Purification Using Color Proteins

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-d-n.png)

After successfully obtaining Curlis, the next step is to acquire target proteins with Spycatcher and verify their binding with Spytag[8], ultimately validating their feasibility for cellulose binding.

We used the J23119 strong promoter to drive the expression of functional proteins, with a His tag fused at the 5' end for downstream purification, and a Spycatcher fused at the 3' end for binding with Spytag. Additionally, a BsaI restriction site was introduced between the His tag and Spycatcher to simplify the integration of other proteins in the future. This allows for seamless assembly into our system through a simple Golden Gate reaction, eliminating the need for tedious molecular cloning.

![](https://static.igem.wiki/teams/5461/engineering/figure-5.jpg)

*Figure5. Schematic Diagram of the Plug-and-Play System*

As a demonstration to validate this system, we selected two commonly used fluorescent and color proteins, sfGFP and amilCP. These proteins were linked to Spycatcher via a linker to construct fusion proteins, which were then inserted into the pCDuet vector. The T7 expression system on the pCDuet was replaced with the J23119 promoter and BBa\_B0034 expression system.

This design allows us to express fluorescent and color proteins with the Spycatcher tag, making it easier to observe the expression of the target sample system and subsequent binding. Additionally, inspired by the blue-white screening experiment, as the target vector for future protein expression vector construction, the colored empty vector provides an effective negative control for the assembly of other target proteins

![](https://static.igem.wiki/teams/5461/engineering/figure-6.png)

*Figure6. Structure of expression Plasmid a. sfGFP expression plasmid b. Amilcp expression plasmid*

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-b-n.png)

All plasmids were obtained through full synthesis and transformed into BL21(DE3). Three single colonies were picked and cultured overnight in LB medium with the corresponding antibiotics. Part of the bacterial culture was sent to Anshengda for sequencing to confirm the correct sequence, while the rest was stored in 30% glycerol for future use.

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-t-n.png)

Colonies were streaked from the glycerol stock, and single colonies were picked and cultured in 50 mL LB medium with streptomycin, incubated overnight at 37°C. The bacterial culture was collected at 10,000 rpm in a 50 mL centrifuge tube and washed with 50 mL of 1X PBS solution. Then, using Thermo B-PER reagent, the bacteria were lysed by sonication, and subsequent purification was performed according to the protocol of the Beyotime His-Tag Purification Kit.

![](https://static.igem.wiki/teams/5461/engineering/figure-7.png)

*Figure7. Expression, Purification, and Identification of Target Proteins.
a. The culture with amilCP (left) appears dark blue, while the culture with sfGFP (right) appears yellow-green.
b. After centrifugation, the sfGFP pellet is yellow-green, and the amilCP pellet is dark blue.
c. The SDS-PAGE results show successful expression of target proteins, with purified proteins being relatively clean. From left to right: total cell lysate with sfGFP plasmid (Lane 1), supernatant sample (Lane 2), purified sample (Lane 3), total cell lysate with amilCP plasmid (Lane 4), supernatant sample (Lane 5), purified sample (Lane 6), and protein molecular weight marker (Lane 7).*

Next, we performed Western blot (WB) analysis on the purified target protein samples. Our target protein carries a His tag, and the target protein bands were detected using an anti-His tag antibody. The results are shown in the following figure:

![](https://static.igem.wiki/teams/5461/engineering/figure-8.png)

*Figure 8. Identification of the Expression of Fluorescent/Color Proteins with Fusion Tags
a. Sample 1: Single colony strain 1 with sfGFP, Control: Negative control group
b. Sample 1: Single colony strain 1 with amilCP, Control: Negative control group*

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-l-n.png)

Through our experimental validation, the plasmids we designed successfully expressed the GFP-SpyCatcher and amilCP-SpyCatcher fusion proteins in Escherichia coli. We completed protein purification and obtained purified products. SDS-PAGE results showed that the molecular weights of the fusion proteins GFP-SpyCatcher and amilCP-SpyCatcher were 40.38 kDa and 39.38 kDa, respectively. However, the molecular weight bands for individual GFP (26.8 kDa), amilCP (25.8 kDa), and SpyCatcher (12.58 kDa) did not appear on the gel, indicating that no monomeric proteins were expressed in our experiment, confirming the successful expression and purification of the fusion proteins. Additionally, the presence of the SpyCatcher peptide did not affect the properties of the color proteins, further enhancing the feasibility of our system.

Next, we are preparing to conduct fusion experiments between the two modules.

#### Cycle 2: Attempt with Antimicrobial Peptide

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/2-d.png)

After successfully engineering the color proteins, we aim to test the assembly and expression of other proteins. We selected a functional antimicrobial peptide as our target protein. Through literature review, we found that antimicrobial peptides can bind well to cellulose, making them suitable for use in antimicrobial dressings for medical applications. After further research, we identified an antimicrobial peptide, HBCM2 (BBa\_K4143336), that can be expressed in Escherichia coli[9]. We decided to encode this peptide upstream of Spycatcher, replacing the color proteins used in the previous demonstration experiment.

![](https://static.igem.wiki/teams/5461/engineering/figure-9-new.png)

*Figure9.Structure of HBCM2 expression Plasmid*

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/2-b.png)

The HBCM2 gene, containing BsaI restriction sites, was obtained through gene synthesis and cloned into the pUC57 vector. To switch vectors, we amplified and extracted the HBCM2 gene from the plasmid, and sequencing confirmed the accuracy of the sequence. We then used the Golden Gate reaction to integrate this gene into our expression plasmid, replacing the original sfGFP gene.

After unsuccessful transformation attempts using the common competent cells DH5a, DH10B, and T1, we decided to use EPI400 super-competent cells[10]. These cells can reduce plasmid copy number, lowering the metabolic burden on E. coli caused by antimicrobial peptide expression, thus allowing us to obtain single colonies with the correct target plasmid.

We transformed the Golden Gate product into EPI400 cells, obtained clones, and selected three single colonies to grow overnight in LB medium with the appropriate antibiotic. Part of the bacterial solution was sent to Anshengda for sequencing confirmation, and the rest was stored in 30% glycerol for future use.

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/2-t.png)

The HBCM2 (AMP) glycerol stock and pCDuet were streaked onto streptomycin plates, and three single colonies were picked and cultured overnight in LB. The next day, the whole culture from the overnight growth was analyzed using SDS-PAGE.

![](https://static.igem.wiki/teams/5461/engineering/figure-10.png)

*Figure 10. Whole-Cell SDS-PAGE Analysis of the Strain Carrying HBCM2 (AMP) and the Strain Carrying the pCDuet Empty Vector.*

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/2-l.png)

1. At the molecular level, our standardized interface design works effectively, allowing us to replace the current fluorescent protein gene with other genes.
2. The presence of Spycatcher does not reduce the burden of antimicrobial peptide expression on bacterial growth. For this class of challenging proteins or those that affect *E. coli* growth, the design of additional molecular chaperones may be needed to assist in their expression.
3. Due to time constraints, we were unable to complete more experiments.

## Optimization of BC Production (Module I)

#### Cycle 1: Improvement of Nitrogen Source for Cellulose Production

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-d-n.png)

A significant challenge in the application of bacterial cellulose (BC) is its high cost. The commonly used HS medium typically consists of 20 g/L glucose, 5 g/L yeast extract, and 5 g/L soytone. These expensive components are difficult to use in large-scale industrial applications. Some studies on alternative carbon sources have discussed replacing glucose with fructose, mannose, and fruit peels. However, since the final product we synthesize is cellulose, substituting carbon sources directly affects the substrate and may significantly impact the quality of the final product[11].

There has been less focus on nitrogen source alternatives in previous work[12-14], primarily centering around corn steep liquor and laundry wastewater. We aim to analyze the nitrogen source preferences of *Komagataeibacter xylinus* using metabolic modeling and to identify potential alternative nitrogen sources. To this end, we utilized genomic data from the *Komagataeibacter xylinus* strain CGMCC 17276, obtained from the China General Microbiological Culture Collection Center (CGMCC), submitted by East China Normal University.

Using the genome of *Komagataeibacter xylinus* strain CGMCC 17276, we generated a corresponding metabolic model through **CarveMe**and imported the resulting SBML file into **cnapy** for Flux Balance Analysis (FBA). To understand the optimal conditions for bacterial cellulose (BC) production, we utilized **cobrapy** to calculate the theoretical optimal medium that maximizes both growth rate and BC production simultaneously.

Based on our metabolic model analysis, the strain shows a higher demand for certain amino acids, specifically arginine (Arg), asparagine (Asn), threonine (Thr), and lysine (Lys). This suggests that these nitrogen sources play a significant role in supporting both growth and bacterial cellulose (BC) production. Interestingly, our model also indicates that the optimal carbon source for maximizing both growth and BC yield is not the commonly used glucose, but rather fructose.

![](https://static.igem.wiki/teams/5461/engineering/figure-11-1.png)

*Figure11. Screenshot of cnapy with our model*

![](https://static.igem.wiki/teams/5461/engineering/figure-11-2.png)

*Figure12.a.Calculate the exchange reaction of the corresponding culture medium when the maximum production growth sum is obtained.
b.The distribution of total growth and production under mixed conditions of three amino acids at different amino acid concentrations.*

Additionally, we explored the effects of varying concentrations of fructose as the carbon source and different nitrogen source combinations in *cn*apy to examine trends in BC production and growth. By conducting these simulations, we were able to analyze the relationship between production and growth, providing insight into how different nutrient conditions influence both the yield of BC and the overall biomass formation of *K. xylinus*. Our approach aims to uncover potential alternative nitrogen sources that can be used to lower the cost of BC production while maintaining high efficiency in industrial applications.

![](https://static.igem.wiki/teams/5461/engineering/figure-11-3a.png)

*Figure 13.a.The relationship between growth and production.*

![](https://static.igem.wiki/teams/5461/engineering/figure-11-3b.png)

*Figure13.b.The total growth and production at different fructose concentrations.*

![](https://static.igem.wiki/teams/5461/engineering/figure-11-4.png)

*Figure14. The distribution of growth and production efficiency under different nitrogen source combinations.*

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-b-n.png)

We selected three candidate nitrogen sources from non-food crops: cottonseed meal, soybean meal, and corn steep liquor, and analyzed their specific amino acid compositions. Cottonseed meal is rich in arginine, making it an ideal choice to meet this requirement, but its lysine and threonine contents are low, which cannot fully satisfy the demand for these amino acids. Soybean meal has insufficient levels of arginine and asparagine. While corn steep liquor has a more balanced amino acid profile with moderate lysine content, it is relatively low in arginine and asparagine.

This combination balances the strain’s amino acid needs while being cost-effective. We designed an experimental test using a total nitrogen equivalent of 40 mg/L to calculate the final concentration of each nitrogen source. Three groups were set up: one using only corn steep liquor, one using only cottonseed meal, and one with a cottonseed meal to corn steep liquor ratio of 1:1. Soytone was used as a positive control. The experiment was conducted in a 250 mL, 10x16 cm stainless steel pan culture system, with three replicates per group for experimental validation.

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-t-n.png)

We incubated both plates statically at 30°C for 5 days, during which we monitored the formation of cellulose mats. At the end of the incubation period, we harvested the BC mats, washed them thoroughly with distilled water, and treated them with 4M NaOH at 80°C for 10min to remove residual cells. After neutralizing with water, we dried the cellulose mats in an oven at 80°C until constant weight was achieved.

To calculate the conversion efficiency of bacterial cellulose (BC) production, we used the formula:

Conversion Efficiency (%) = (Dry Weight of BC Produced / Total Sugar Input) x 100

For each experimental condition, we measured the dry weight of the BC mats after drying to constant weight and compared this to the total mass of nitrogen sources used in the culture medium. For example, if 2.5 grams of BC were produced using 5 grams of Sugar, the conversion efficiency would be 50%.

Our findings demonstrated that the original soytone condition provided the highest conversion efficiency for BC production, with the mixture of corn steep liquor and cottonseed meal yielding higher conversion rates than either alternative used alone. This study provides insight into the potential of using cost-effective, alternative nitrogen sources for the production of bacterial cellulose while maintaining high efficiency.

![](https://static.igem.wiki/teams/5461/engineering/figure-12.png)

*Figure 15. Comparison of BC Yield under Different Nitrogen Sources*

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-l-n.png)

Our experiment shows that, for now, cottonseed meal and soybean meal alone cannot fully replace soytone. However, combining cottonseed meal with soybean meal or corn steep liquor can compensate for the deficiencies of a single nitrogen source. This combination meets the demand for arginine, supplements lysine and threonine, and helps control production costs. This approach may potentially be used in the future to reduce the use of soytone.

#### Cycle2 : Improvement of Cellulose Cultivation Environment

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/2-d.png)

In the previous experiment, we observed an interesting phenomenon: the thickness of the BC membrane we cultivated was generally around 0.5 cm, but there was still some remaining liquid medium underneath it. We hypothesize that this is because the liquid below is unable to access oxygen, and BC is only produced at the interface between oxygen and the liquid. To optimize bacterial cellulose production efficiency, we designed an experiment to study the effect of culture vessel size on production efficiency while keeping the volume of the medium constant.

We selected two different culture plate sizes: a smaller 10 x 16 cm plate and a larger 15 x 27 cm plate. We hypothesize that the larger plate's greater surface area will enhance gas exchange and nutrient distribution, thereby improving cellulose production efficiency. By comparing the cellulose yield from these two different plate sizes, we aim to determine whether the size of the container can significantly impact conversion rates and overall production efficiency. This experimental design allows us to explore how physical dimensions affect key factors in bacterial cellulose synthesis.

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/2-b.png)

Each plate was filled with 250 mL of HS media, which we prepared by dissolving 20 g of glucose, 5 g of peptone, 5 g of yeast extract, 2.7 g of disodium phosphate, and 1.15 g of citric acid per liter of distilled water, followed by autoclaving. After the media cooled, we inoculated both plates with the bacterial strain Komagataeibacter xylinus, ensuring equal inoculum concentration and uniform distribution across the surface of the media.

![](https://static.igem.wiki/teams/5461/engineering/figure-13.png)

*Figure 16. Experimental Results of BC Production in Different Containers*

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/2-t.png)

We incubated both plates statically at 30°C for 5 days, during which we monitored the formation of cellulose mats. At the end of the incubation period, we harvested the BC mats, washed them thoroughly with distilled water, and treated them with 4M NaOH at 80°C to remove residual cells. After neutralizing with water, we dried the cellulose mats in an oven at 80°C until constant weight was achieved. We then weighed each replicate individually and performed statistical analysis and plotting.

Our results showed that the smaller plate (10 x 16 cm) resulted in a conversion rate of 11.5% on average, while the larger plate (15 x 27 cm) achieved a conversion rate of over 20%. We believe that the larger surface area of the 15 x 27 cm plate allowed for better gas exchange and nutrient distribution, leading to more efficient cellulose production. This experiment demonstrates that the dimensions of the cultivation container play a critical role in bacterial cellulose production efficiency, even when the media volume remains constant.

![](https://static.igem.wiki/teams/5461/engineering/figure-14.png)

*Figure 17. Bar Chart Comparing BC Yield in Different Containers*

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/2-l.png)

From the experimental results, we can learn that the surface area of the culture vessel has a significant impact on the efficiency of bacterial cellulose production. The larger culture plate (15 x 27 cm), with its greater surface area, facilitated better gas exchange and nutrient distribution, resulting in a higher cellulose conversion rate. Therefore, future designs should prioritize increasing the culture surface area while optimizing gas exchange and nutrient distribution, or explore different vessel shapes to enhance production efficiency. The experiment also showed that even with the culture medium volume remaining constant, changes in container size significantly affect production outcomes. This provides quantitative guidance for container selection in large-scale production.

## Composition: Modules Composition Experiment

#### Cycle 1: Three-Module Composition Verification

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-d-n.png)

We designed a control experiment to verify the binding of bacterial cellulose, Curlis-Spytag, and color protein-Spycatcher.

![](https://static.igem.wiki/teams/5461/results/table.png)

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-b-n.png)

1. Cut a piece of BC gel into small blocks of similar size.

2. Take 6 stainless steel bowls (304 grade), and add 50 ml of ddH2O, 50 ml of POI-Spycatcher solution, and 50 ml of POI-Spycatcher-Curlis-Spytag binding solution into each. Place the similarly sized gel blocks into the solutions and incubate overnight at room temperature with shaking at 80 rpm.

![](https://static.igem.wiki/teams/5461/engineering/figure-15-small.png)

*Figure 18. Experimental Setup and Process for Composition Analysis*

3. The next day, remove the corresponding cellulose from the stainless steel bowls and place it in 50 ml ddH2O. Incubate at room temperature with shaking at 80 rpm for 6 hours.

4. Remove the gel from each group, drain, and take photos.

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-t-n.png)

![](https://static.igem.wiki/teams/5461/results/figure-9-new-1.png)

*Figure 19. Comparison of Different Experimental Conditions for Bacterial Cellulose Binding with AmilCP*

![](https://static.igem.wiki/teams/5461/results/figure-10-new-1.png)

Figure 20. Comparison of Different Experimental Conditions for Bacterial Cellulose Binding with sfGFP

###### ![Icon](https://static.igem.wiki/teams/5461/icon/dbtl/1-l-n.png)

***Figure 19 and Figure 20*** show the experimental comparison of amilCP and sfGFP binding to bacterial cellulose via scaffold proteins under different conditions. In both experiments, it was observed that the experimental groups with the mixed scaffold protein solution exhibited a deeper color compared to the groups without the scaffold protein solution. This indicates that our scaffold protein design enhances the binding capability between the POI (Protein of Interest) and bacterial cellulose.

## Work Going on

1. We aim to identify additional protein pairs with strong binding affinity, similar to the SpyTag-SpyCatcher system, to accommodate a wider range of proteins for our scaffold protein work.
2. For antimicrobial peptides, iGEM22\_Michigan has provided an effective strategy by using a molecular chaperone to enhance the expression of antimicrobial peptides in *E. coli*. We plan to adopt this approach to validate the expression of our antimicrobial peptide-SpyCatcher system, integrating it into our platform. This will allow for future compatibility with proteins that impose a high metabolic burden on bacterial growth.

## Referrence

1. Avcioglu, N.H., 2022. Bacterial cellulose: recent progress in production and industrial applications. World Journal of Microbiology and Biotechnology, 38(5), p.86. Published on 10 April 2022. doi:10.1007/s11274-022-03271-y.
2. Cielecka, I., Ryngajłło, M., Maniukiewicz, W. & Bielecki, S., 2021. Highly stretchable bacterial cellulose produced by Komagataeibacter hansenii SI1. Polymers, 13(24), p.4455. Published on 19 December 2021. doi:10.3390/polym13244455.
3. [https://2014.igem.org/Team:Imperial/Project\_Background](https://2014.igem.org/Team%3AImperial/Project_Background)
4. [https://2017.igem.org/Team:TUST\_China/Description](https://2017.igem.org/Team%3ATUST_China/Description)
5. [https://2020.igem.org/Team:JNFLS/Design](https://2020.igem.org/Team%3AJNFLS/Design)
6. [https://2021.igem.org/Team:SZPT-CHINA/Design](https://2021.igem.org/Team%3ASZPT-CHINA/Design)
7. Khatri, V., Jafari, M., Gaudreault, R., et al., 2023. Bionanocomposites with enhanced physical properties from curli amyloid assemblies and cellulose nanofibrils. Biomacromolecules, 24(11), pp.5290-5302. doi:10.1021/acs.biomac.3c00786.
8. Hatlem, D., Trunk, T., Linke, D. & Leo, J.C., 2019. Catching a SPY: using the SpyCatcher-SpyTag and related systems for labeling and localizing bacterial proteins. International Journal of Molecular Sciences, 20(9), p.2129. Published on 30 April 2019. doi:10.3390/ijms20092129.
9. Lee, T.H., Carpenter, T.S., D'haeseleer, P., Savage, D.F. & Yung, M.C., 2020. Encapsulin carrier proteins for enhanced expression of antimicrobial peptides. Biotechnology and Bioengineering, 117(3), pp.603-613. doi:10.1002/bit.27222.
10. Grefen, C., et al., 2008. Subcellular localization and in vivo interactions of the Arabidopsis thaliana ethylene receptor family members. Molecular Plant, 1, pp.308-320.
11. Rouhi, M., Khanchezar, S. & Babaeipour, V., 2023. Economical optimization of industrial medium culture for bacterial cellulose production. Applied Biochemistry and Biotechnology, 195(5), pp.2863-2881. doi:10.1007/s12010-022-04239-2.
12. Fernandes, I.A.A., Pedro, A.C., Ribeiro, V.R., et al., 2020. Bacterial cellulose: from production optimization to new applications. International Journal of Biological Macromolecules, 164, pp.2598-2611. doi:10.1016/j.ijbiomac.2020.07.255.
13. Avcioglu, N.H., 2022. Bacterial cellulose: recent progress in production and industrial applications. World Journal of Microbiology and Biotechnology, 38(5), p.86. Published on 10 April 2022. doi:10.1007/s11274-022-03271-y.
14. Bae, S. & Shoda, M., 2005. Statistical optimization of culture conditions for bacterial cellulose production using Box-Behnken design. Biotechnology and Bioengineering, 90(1), pp.20-28. doi:10.1002/bit.20325.

{% endblock %}

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block cover %}

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# Experiments

{% endblock %}
{% block page\_content %}

![Cover Image](https://static.igem.wiki/teams/5461/page-first-img/experiment-new.jpg)

Experiments

![](https://static.igem.wiki/teams/5461/icon/fen.png)Stock Solution Preparation
![](https://static.igem.wiki/teams/5461/icon/fen.png)LB Medium Preparation
![](https://static.igem.wiki/teams/5461/icon/fen.png)LB Agar Plate Preparation
![](https://static.igem.wiki/teams/5461/icon/fen.png)Congo Red Plate Preparation
![](https://static.igem.wiki/teams/5461/icon/fen.png)Bacterial Transformation
![](https://static.igem.wiki/teams/5461/icon/fen.png)Protein Expression
![](https://static.igem.wiki/teams/5461/icon/fen.png)POI Purification and Characterization
![](https://static.igem.wiki/teams/5461/icon/fen.png)Curlis Fiber Purification and Characterization
![](https://static.igem.wiki/teams/5461/icon/fen.png)Congo Red Characterization of Curlis Fiber
![](https://static.igem.wiki/teams/5461/icon/fen.png)SDS-PAGE
![](https://static.igem.wiki/teams/5461/icon/fen.png)Binding Assay Verification

![](https://static.igem.wiki/teams/5461/icon/zi.png)Back to the top

## Stock Solution Preparation

1. #### 50 mg/ml Kanamycin Stock Reagent

   1. 1 g kanamycin powder (Beyotime: ST101) was weighed and added to a 50 ml centrifuge tube. Subsequently, 20 ml of sterilized ddH2O (Sangon Biotech: B541017) was added. The mixture was vortexed to homogenize, allowed to stand for 3 minutes, and then sterilized by filtration using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). The resulting 20 ml of 50 mg/ml kanamycin stock solution was aliquoted into 2 ml EP tubes for storage.
2. #### 50 mg/ml Streptomycin Stock Reagent

   1. 500 mg streptomycin powder (Beyotime: ST2722-1g) was weighed and transferred into a 50 ml centrifuge tube. A volume of 10 ml of sterilized ddH2O (Sangon Biotech: B541017) was then added. The mixture was vortexed, allowed to stand for 3 minutes, and subsequently sterilized by filtration using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). The resulting 10 ml of 50 mg/ml streptomycin stock solution was aliquoted into 2 ml EP tubes for storage.
3. #### 100 mM IPTG Stock Reagent

   * A 1.2 g quantity of IPTG powder (Beyotime: ST098) was weighed and added to a 50 ml centrifuge tube. The tube was then filled with 50 ml of sterilized ddH2O (Sangon Biotech: B541017). The solution was vortexed, allowed to stand for 3 minutes, and filtered for sterilization using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). The resulting 50 ml of 100 mM IPTG stock solution was aliquoted into 2 ml EP tubes for storage.
4. #### 10 mg/ml Congo Red Stock Reagent

   1. 0.5 g quantity of Congo Red powder (Aladdin: C196465) was weighed and transferred into a 50 ml centrifuge tube. The tube was filled with 50 ml of sterilized ddH2O (Sangon Biotech: B541017). The solution was vortexed to mix thoroughly, allowed to stand for 3 minutes, and sterilized by filtration using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). The resulting 50 ml of 10 mg/ml Congo Red stock solution was aliquoted into 2 ml EP tubes for storage.
5. #### 10 mg/ml Coomassie Brilliant Blue Stock Reagent

   1. 0.5 g quantity of Coomassie Brilliant Blue G-250 powder (Beyotime: ST1119) was weighed and transferred into a 50 ml centrifuge tube. The tube was filled with 50 ml of sterilized ddH2O (Sangon Biotech: B541017). The mixture was vortexed, allowed to stand for 3 minutes, and sterilized by filtration using a 0.22 μm sterile membrane filter (Merck Millex™-GS Sterile Syringe Filter Unit, MCE). The resulting 50 ml of 10 mg/ml Coomassie Brilliant Blue stock solution was aliquoted into 2 ml EP tubes for storage.

## LB Medium Preparation

1. A bottle of BeyoPure™ LB Broth (Catalog No. ST156) was opened and the entire contents were added to a 1L blue-capped bottle. Ultrapure water from the Milli-Q® Type 1 Ultrapure Water System was added to a total volume of 500 ml. The mixture was shaken well, autoclave tape was applied, the bottle cap was loosened, and the solution was sterilized at 121°C for 30 minutes.

## LB Agar Plate Preparation

1. A bottle of BeyoPure™ LB Broth with Agar (Catalog No. ST158) was opened and the entire contents were added to a 1L blue-capped bottle. Ultrapure water from the Milli-Q® Type 1 Ultrapure Water System was added to a final volume of 500 ml. The solution was shaken thoroughly, autoclave tape was applied, the bottle cap was loosened, and the solution was sterilized at 121°C for 30 minutes. After cooling to approximately 60°C, the corresponding antibiotic was added.

## 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 |

## Bacterial Transformation

1. Trans BL21(DE3) Chemically Competent Cells (TransGene CD601-02) were retrieved from the -80°C freezer and allowed to thaw on ice for 10 minutes.
2. 10 ng of the target plasmid was added to the thawed competent cells, gently mixed, and incubated on ice for 30 minutes. Meanwhile, a water bath was prepared at 42°C.
3. The competent cell-plasmid mixture was subjected to heat shock at 42°C for 45 seconds, followed by an immediate transfer to ice for 2 minutes without agitation of the centrifuge tube.
4. Subsequently, 500 µl of sterile SOC medium was added to each tube, mixed thoroughly, and incubated at 37°C with shaking at 200 rpm for 1 hour to facilitate bacterial recovery. Meanwhile, LB plates with the appropriate antibiotic were prepared and placed in the 37°C incubator.
5. Transformed competent cells were plated onto LB agar plates containing the appropriate antibiotic and spread evenly. The plates were incubated at 37°C until the liquid was absorbed, then inverted and incubated overnight at 37°C.

![](https://static.igem.wiki/teams/5461/freeze/experiments-transformation.png)

A glimpse of our wet-lab experiments 1

## Protein Expression

1. #### POI (Protein of Interest) Expression

   1. 3 ml volume of LB medium with the appropriate antibiotic was added to a 12 ml culture tube.
   2. Three single colonies were selected from the plate and inoculated into the 3 ml LB medium with the appropriate antibiotic.
   3. The culture was incubated overnight in a shaker at 220 rpm and 37°C.
2. #### Curlis Fiber Expression

   1. 3 ml volume of LB medium with the appropriate antibiotic was added to a 12 ml culture tube.
   2. Three single colonies were selected from the plate and inoculated into the 3 ml LB medium with the appropriate antibiotic.
   3. The culture was incubated overnight in a shaker at 220 rpm and 37°C.
   4. After overnight incubation, 40 µl overnight culture was transferred into 4 ml of LB medium with the appropriate antibiotic and incubated in a shaker at 220 rpm and 37°C for 2 hours. 40 µl 100mM IPTG was added to a final concentration of 1 mM, and the culture was incubated at 220 rpm and 30°C for 36 hours.
   5. After overnight incubation, filamentous precipitates were observed in the culture medium. The precipitates were collected using an inoculating loop and dissolved in 4M guanidine hydrochloride.

## POI Purification and Characterization

1. The cells were centrifuged at 10000 rpm for 5 minutes, and the supernatant was discarded.
2. A volume of 8 ml of denaturing lysis buffer was added, and the mixture was homogenized.
3. The lysate was subjected to ultrasonic disruption for 5 minutes under the following conditions:
   1. Power: 600W
   2. Sonication on-time: 5 seconds
   3. Sonication off-time: 5 seconds
4. The lysate was centrifuged, and the supernatant was collected for further processing.
   1. Protein purification was carried out using the Beyotime His-tag Protein Purification Kit (Denaturant-resistant; P2229S) or Beyotime GST-tag Protein Purification Kit (Denaturant-resistant; P2262).
      1. The BeyoGold™ 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.

## Curlis Fiber Purification and Characterization

1. The filamentous precipitates were collected from the culture medium using an inoculating loop or pipette tip and dissolved in 4M guanidine hydrochloride. The mixture was homogenized until fully dissolved, serving as the supernatant for subsequent purification.
   1. Protein purification was carried out using the Beyotime His-tag Protein Purification Kit (Denaturant-resistant; P2229S).
      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.
      6. SDS-PAGE analysis was conducted to verify the purity of the eluted protein.

## Congo Red Characterization of Curlis Fiber

1. The correctly sequenced Curlis Fiber-pET28a and empty pET28a glycerol stocks were retrieved from the -80°C freezer, streaked onto kanamycin plates, and incubated overnight at 37°C. After overnight incubation, the plates were stored at 4°C for up to one week.
2. Three single colonies were selected from the plate and inoculated into 3 ml of LB medium with kanamycin, followed by incubation at 220 rpm and 37°C overnight.
3. 30 µl overnight culture was spotted onto Congo Red plates containing the appropriate antibiotic and different concentrations of IPTG. After the spots dried, the plates were inverted and incubated at 30°C in a controlled environment for 48 hours.
4. Photographs were taken to document the results.

## SDS-PAGE

1. 20 µl aliquot of each sample was mixed with 5 µl of SDS-PAGE protein loading buffer (Boyetime P0015), vortexed, and incubated at 98°C in a water bath for 10 minutes to prepare the samples for loading.
2. The samples were centrifuged at 10000 rpm for 1 minute.
3. 1X MOPS-SDS running buffer was prepared by diluting 50 ml of 20X MOPS-SDS running buffer in 950 ml of ddH2O.
4. YoungPAGE™ (Genscript: M00928) pre-cast gels were set up in a Biorad Mini-PROTEAN Tetra Vertical Electrophoresis Cell according to the manufacturer’s instructions, and the 1X MOPS-SDS running buffer was added.
5. 10 µl of the prepared samples was loaded into the gel wells, and Broad Multi Color Pre-Stained Protein Standard (Genscript: M00624) was added as a protein molecular weight marker in the empty wells.
6. Electrophoresis was performed at 200 mV for 20 minutes, with the electrophoresis time adjusted as needed based on the results.
7. Upon completion of electrophoresis, the SDS gel was removed and subjected to staining and destaining procedures as per the manufacturer’s instructions.
8. After destaining, the gel was placed in a ChemiDoc Imaging System (Biorad: 12003153) for imaging, and the images were uploaded.

## Binding Assay Verification

1. Equal volumes (50 µl) of curlis fiber and POI protein solutions were mixed and incubated overnight at 30°C with shaking at 80 rpm.
2. SDS-PAGE was performed to verify the binding interactions.

![](https://static.igem.wiki/teams/5461/freeze/experiments-wetlab.png)

A glimpse of our wet-lab experiments 2

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# Results

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![Cover Image](https://static.igem.wiki/teams/5461/page-first-img/results2.jpg)

Results

## Results: Proof of Concept

Design and Experimental Validation of a Modular System for Efficient Bacterial Cellulose Composite Modification

**Bacterial cellulose**, as a natural high molecular material, possesses abundant resources, renewability, and **biocompatibility**, making it highly promising for applications across various fields. To address the challenge of bacterial cellulose modification, we have developed a modular approach to bacterial cellulose composite modification. This system aims to **streamline** and enhance the efficiency of the modification process by employing protein-bacterial cellulose combination. The system consists of three core modules, we validated the following three modules and the composition through experiments.

![](https://static.igem.wiki/teams/5461/icon/fen.png)Overview
![](https://static.igem.wiki/teams/5461/icon/fen.png)Module I: BC
![](https://static.igem.wiki/teams/5461/icon/fen.png)Module II: Scaffold
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Congo Red Staining Experiment
![](https://static.igem.wiki/teams/5461/icon/cheng.png)Expression and Purification in Liquid
![](https://static.igem.wiki/teams/5461/icon/fen.png)Module III: Fusion Protein
![](https://static.igem.wiki/teams/5461/icon/fen.png)Composition
![](https://static.igem.wiki/teams/5461/icon/zi.png)Back to the top

## Overview: Bacterial Cellulose Modification System

The following illustration shows the architecture of our Bacterial Cellulose Modification System:

![](https://static.igem.wiki/teams/5461/engineering/architecture.jpg)

*Bacterial Cellulose Modification System*

#### Module I. Bacterial Cellulose Production

*Komagataeibacter* is a microorganism widely utilized in large-scale bacterial cellulose production. We have optimized the production process, particularly by refining nitrogen source selection and cultivation conditions, thereby significantly improving both the yield and quality of the produced bacterial cellulose.

#### Module II. Scaffold Protein Design and Production

Through genetic engineering, we successfully constructed an *Escherichia coli* strain that expresses a scaffold protein, **Curlis Fiber-Spytag**, capable of binding to bacterial cellulose. In this system, the Curlis Fiber domain provides the bacterial cellulose-binding functionality, while **Spytag** enables the heterologous linkage via the SpyCatcher system.

#### Module III. Target Protein Design and Production

We designed a ***SpyCatcher-POI* (protein of interest) fusion** with a standardized interface for expression and purification. Using two distinct color proteins as demonstration models, we validated both the expression and purification processes. In future applications, we anticipate that this approach will allow the **facile substitution of various POIs** through the Golden Gate cloning method.

#### Composition

Finally, our experimental results confirmed the high-efficiency binding of **bacterial cellulose**, the scaffold protein ***Curlis Fiber-Spytag***, and the target protein ***SpyCatcher-POI***.

These findings demonstrate the **effectiveness and feasibility of this modular system**, providing a POC and novel strategy for the modification and application of bacterial cellulose.

![](https://static.igem.wiki/teams/5461/freeze/engineering-wet-lab.png)

*A Glimpse of our wet-lab work*

## Module I: Optimization of Conditions for BC Production

**Impact of Nitrogen Source and Cultivation Container Size on Bacterial Cellulose Production Efficiency**

We utilized the Komagataeibacter xylinus strain CGMCC 17276, obtained from the China General Microbiological Culture Collection Center (CGMCC), submitted by East China Normal University, to explore the impact of alternative nitrogen sources on bacterial cellulose (BC) production. The genome-scale metabolic model (GEM) for this strain was constructed using the CarveMe pipeline, and simulations were performed in the CobraPy environment. Growth media formulations were based on standard Lysogeny Broth (LB) and Hestrin-Schramm (HS) media, with soytone serving as the original nitrogen source[*Figure 1*].

![](https://static.igem.wiki/teams/5461/results/figure-1-new.png)

*Figure1.The distribution of growth and production efficiency under different nitrogen source combinations.(for details, refer to the Engineer Page)*

We aimed to reduce production costs by substituting soytone with alternative, non-food biomass nitrogen sources, specifically corn steep liquor and cottonseed meal. The nitrogen sources were tested individually and in equal mixtures, while maintaining the total nitrogen concentration equivalent to the nitrogen content in 5 g/L soytone. Using the GEM, we simulated the impact of these nitrogen sources on BC production and validated the results in a 250 mL HS media system, cultivated in 10 x 16 cm stainless steel plates. To calculate the conversion efficiency of bacterial cellulose (BC) production, we used the formula:
Conversion Efficiency (%)=(Dry Weight of BC Produced / Total Sugar Input )×100

*Figure2* demonstrated that the original soytone condition provided the highest conversion efficiency for BC production, with the mixture of corn steep liquor and cottonseed meal yielding higher conversion rates than either alternative used alone. This study provides insight into the potential of using cost-effective, alternative nitrogen sources for the production of bacterial cellulose while maintaining high efficiency.

![](https://static.igem.wiki/teams/5461/results/figure-2-new.png)

*Figure2: Exploration of the Impact of BC Cultivation Containers*

We conducted an experiment to investigate how the dimensions of stainless steel plates affect the conversion rate of bacterial cellulose (BC) production. Using the same volume of Hestrin-Schramm (HS) media, we compared two stainless steel plates: one measuring 10 x 16 cm and the other 15 x 27 cm. Each plate was filled with 250 mL of HS media, which we prepared by dissolving 20 g of glucose, 5 g of peptone, 5 g of yeast extract, 2.7 g of disodium phosphate, and 1.15 g of citric acid per liter of distilled water, followed by autoclaving. After the media cooled, we inoculated both plates with the bacterial strain \*Komagataeibacter xylinus\*, ensuring equal inoculum concentration and uniform distribution across the surface of the media.

We incubated both plates statically at 30°C for 5 days, during which we monitored the formation of cellulose mats. At the end of the incubation period, we harvested the BC mats, washed them thoroughly with distilled water, and treated them with 2M NaOH at 80°C to remove residual cells. After neutralizing with water, we dried the cellulose mats in an oven at 80°C until constant weight was achieved.To calculate the conversion efficiency of bacterial cellulose (BC) production, we used the formula:
Conversion Efficiency (%)=(Dry Weight of BC Produced / Total Sugar Input )×100

*Figure 3* showed that the smaller plate (10 x 16 cm) resulted in a conversion rate of 11.5% on average, while the larger plate (15 x 27 cm) achieved a conversion rate of over 20%. We believe that the larger surface area of the 15 x 27 cm plate allowed for better gas exchange and nutrient distribution, leading to more efficient cellulose production. This experiment demonstrates that the dimensions of the cultivation container play a critical role in bacterial cellulose production efficiency, even when the media volume remains constant.

![](https://static.igem.wiki/teams/5461/results/figure-2-new.png)

*Figure3: BC conversion rate from Gluc in different plates*

## Module II: Production & Purification of Curlis-Spytag (Scaffold)

#### 1.Congo Red Staining Experiment

Congo Red Staining Confirms Expression and Self-Assembly of Curlis-Spytag Protein.[*Figure 4*]

We used the Congo Red staining experiment to detect the expression and self-assembly of Curlis-Spytag protein. Strains expressing *curlis fiber* and strains carrying the empty pET28a+ vector were separately plated on Congo Red plates and control plates with 0mM, 0.5mM, and 1mM IPTG. We pipetted 30 μL of overnight culture onto plates with varying concentrations of Congo Red and control plates, then incubated them at 30°C for 48 hours to observe changes in colony color and self-assembly.

We observed that on the Congo Red plates containing 0.5mM and 1mM IPTG, the strain expressing curlis fiber exhibited a pink color (colonies on the sides), while the strain with the empty pET28a+ vector (middle colony) remained white. Based on these results, we preliminarily conclude that the *curlis fiber*-expressing strain is capable of expressing CsgA, secreting it extracellularly, and assembling into curlis fibers, which appear pink in the presence of Congo Red. The same strain on control plates containing 0.5mM and 1mM IPTG appeared entirely white, indicating that the results observed on the Congo Red plates are specific.

![](https://static.igem.wiki/teams/5461/results/figure-4-new.png)

*Figure 4. Congo Red staining experiment detecting the expression and self-assembly of Curlis-Spytag protein. The growth conditions of strains expressing curlis fiber and strains carrying the empty pET28a+ vector on Congo Red plates with 0mM, 0.5mM, and 1mM IPTG (upper panel) and control plates (lower panel) are shown.*

#### 2.Expression and Purification in Liquid

Successful Expression, Purification, and Detection of Curlis-Spytag Fibers via Western Blot

We took samples from the glycerol stock of Curlis-Spytag and streaked them onto LB agar plates containing kanamycin to isolate single colonies. Simultaneously, we streaked pET28a+ empty vector colonies as controls onto LB agar plates containing kanamycin. From the agar plates, we selected three Curlis-Spytag single colonies and inoculated them into LB liquid medium containing kanamycin, followed by overnight incubation at 37°C, 220 rpm. On the second day, we diluted the overnight cultures 1:100 into fresh LB medium and induced the cultures with IPTG at different concentrations when the OD600 reached 0.3. After induction, the cultures were incubated at 30°C, 220 rpm for another 24 hours. After 24 hours, we observed significant fibrous precipitates in liquid cultures, indicating that the Curlis fibers. were successfully expressed and self-assembled into fibrous structures[*Figure 5*].

![](https://static.igem.wiki/teams/5461/results/figure-5-new.png)

*Figure 5. Self-assembly of Curlis Fibers in Liquid Culture Medium
a. Growth status of bacterial strains expressing Curlis fibers and the filamentous precipitates (Curlis fibers) in liquid culture medium with 0mM, 0.1mM, and 1mM IPTG concentrations.
b. Growth status of the strain carrying the empty pET28a+ plasmid, and two single colonies expressing Curlis fibers, as well as the filamentous precipitates (Curlis fibers), in liquid culture medium with 0mM, 0.1mM, and 1mM IPTG concentrations.*

After washing with ddH2O, 1X PBS, and 5X PBS, we discovered that the fibrous precipitates in the culture could not dissolve uniformly. To dissolve the Curlis fibers and detect the expression of Curlis through electrophoresis, we decided to use 8M urea and eventually succeeded in dissolving the fibrous precipitates. We then used the GST purification kit from Beyotime to purify the protein solution after dissolution in 8M urea, removing nonspecific proteins and obtaining high-purity Curlis target proteins. The purified Curlis protein samples were then analyzed using Western Blot (WB). The designed Curlis-Spytag fusion protein carried a GST tag, and we were able to detect the target protein bands using anti-GST tag antibodies.*[Figure 6]*

![](https://static.igem.wiki/teams/5461/results/figure-6-new.png)

*Figure 6. Identification of the Expression of Curlis Protein with Fusion Tag
Sample 1: Curlis-Spytag single colony strain No.1, Control: Negative control group.*

The results confirmed that the Curlis-Spytag fibers could be efficiently dissolved in 8M urea into a soluble state, and the fibrous precipitates obtained from two independent colonies could be detected in subsequent immunoblot experiments.

## Module III: Production and Purification of POI-SpyCatcher (Fusion Protein)

**Successful Expression and Purification of Fusion Proteins**

We streaked bacteria from glycerol stocks containing the GFP-SpyCatcher and amilCP-SpyCatcher plasmids, selected single colonies, and inoculated them into 50 ml of LB ste liquid medium for overnight incubation at 37°C. The bacterial cultures were collected by centrifugation at 10,000 rpm in 50 ml centrifuge tubes, and the pellets were resuspended and washed with 50 ml of 1X PBS buffer. Next, we used Thermo B-PER reagent to lyse the bacteria through sonication, and then performed protein purification using the His-Tag purification kit from Beyotime. The experimental results demonstrated that the plasmids we designed successfully expressed the GFP-SpyCatcher and amilCP-SpyCatcher fusion proteins in Escherichia coli. SDS-PAGE and Western blotting results showed that the fusion proteins GFP-SpyCatcher and amilCP-SpyCatcher had molecular weights of 40.38 kDa and 39.38 kDa, respectively. However, the molecular weight bands of the individual proteins GFP (26.8 kDa), amilCP (25.8 kDa), and SpyCatcher (12.58 kDa) were not observed on the gel, indicating that no monomeric proteins were expressed in our experiment. This confirmed the successful expression and purification of fusion proteins. Furthermore, the presence of the SpyCatcher peptide did not affect the properties of the color proteins.

![](https://static.igem.wiki/teams/5461/results/figure-7-new.png)

*Figure 7. Expression, Purification, and Identification of Target Proteins.
a. The culture with amilCP (left) appears dark blue, while the culture with sfGFP (right) appears yellow-green.
b. After centrifugation, the sfGFP pellet is yellow-green, and the amilCP pellet is dark blue.
c. The SDS-PAGE results show successful expression of target proteins, with purified proteins being relatively clean. From left to right: total cell lysate with sfGFP plasmid (Lane 1), supernatant sample (Lane 2), purified sample (Lane 3), total cell lysate with amilCP plasmid (Lane 4), supernatant sample (Lane 5), purified sample (Lane 6), and protein molecular weight marker (Lane 7).*

![](https://static.igem.wiki/teams/5461/results/figure-8-new.png)

*Figure 8. Identification of the Expression of Fluorescent/Color Proteins with Fusion Tags
a. Sample 1: Single colony strain 1 with sfGFP, Control: Negative control group
b. Sample 1: Single colony strain 1 with amilCP, Control: Negative control group*

## Composition

We designed a control experiment to verify the binding of bacterial cellulose, Curlis-Spytag, and color protein-Spycatcher.

![](https://static.igem.wiki/teams/5461/results/table.png)

A piece of BC gel is cut into small blocks of similar size. Six stainless steel bowls (304 grade) are prepared, each containing 50 ml of ddH2O, 50 ml of POI-Spycatcher solution, and 50 ml of POI-Spycatcher-Curlis-Spytag binding solution. The similarly sized gel blocks are then placed into the solutions and incubated overnight at room temperature with shaking at 80 rpm. The following day, the cellulose is removed from the stainless steel bowls and placed into 50 ml of ddH2O. This is incubated at room temperature with shaking at 80 rpm for 6 hours. Subsequently, the gel is removed from each group, drained, and photographs are taken.

![](https://static.igem.wiki/teams/5461/results/figure-9-new-1.png)

*Figure9. Comparison of Different Experimental Conditions for Bacterial Cellulose Binding with AmilCP*

![](https://static.igem.wiki/teams/5461/results/figure-10-new-1.png)

*Figure10.Comparison of Different Experimental Conditions for Bacterial Cellulose Binding with sfGFP*

*Figure 9* and *Figure 10* represent the experiments where amilCP and sfGFP were bound to bacterial cellulose via scaffold proteins. A comparative analysis revealed that the experimental groups with mixed scaffold protein solutions exhibited a noticeably deeper color than those without the scaffold proteins. This indicates that our scaffold protein design enhances the binding efficiency between the Protein of Interest (POI) and bacterial cellulose.

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