iGEM 2025 — Team nau-china

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Notebook

"Notebook is a time map for our project, marking where we started and how far we've come." — NAU-CHINA

![Classroom Scene](https://static.igem.wiki/teams/5855/heroimg/notebook.webp)

January - February

March

April

May

June

July

August

September

October

![](https://static.igem.wiki/teams/5855/generalimg/lineline.svg)

![指示器](https://static.igem.wiki/teams/5855/generalimg/mvsz-half.webp)

## January - February

From January 18th to February 8th, led by NAU-CHINA 2024, we held the winter training on synthetic
biology and the iGEM competition. Our winter training was open to all students interested in iGEM and
synthetic biology. The program was designed with the following goals:
1. **Introduction to**
**iGEM** **and synthetic biology:** Students were introduced to the history, rules, and
significance of the iGEM competition, along with the basic principles and core concepts of synthetic
biology. This laid a solid foundation for their further involvement.
2. **Training in literature
review and essential software:** Students learned how to conduct efficient literature searches and
gained hands-on experience with commonly used tools such as SnapGene and other bioinformatics software,
equipping them with the skills to independently explore scientific questions. Also, we provided lessons
and guidance for Model, Art, Wiki and HP groups.

![img](https://static.igem.wiki/teams/5855/notebook/january1.webp)

3. **Project design in** **iGEM:** Through case studies and guided discussions, students
explored how to design an iGEM project, including identifying real-world problems, proposing
engineering-based solutions, and integrating both experimental and human practices
aspects.Members
deepened their understanding of common chassis organisms such as **E. coli** and
cyanobacteria, while also discussing diverse applications of synthetic biology in fields ranging from
environmental protection to biomanufacturing.

![img](https://static.igem.wiki/teams/5855/notebook/january.webp)

## March

**1. Registered the team and confirmed core members.**

After rounds of selection, we are proud to announce
the establishment of the **NAU-CHINA 2025 team**!

👉 [Meet our members here.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27members%27%29%20%7D%7D)

![img](https://static.igem.wiki/teams/5855/notebook/nau.webp)

**2. Wet Lab:**

* Brainstormed and reviewed literature across different fields to explore potential research
  topics.
* Identified several directions of interest and evaluated their feasibility.
* Held the team meeting to allocate responsibilities and share initial expectations.

**3. Human Practices:**

* Participated in SBC: Synthetic Biology Conference & Exhibition.
* Carried out our first education-focused Human Practices activity with Grade 8 students from
  Nanjing University of Science and Technology Affiliated Middle School.

![img](https://static.igem.wiki/teams/5855/notebook/march.webp)

## April

**1. Wet Lab:**

* Brainstorming & Literature Review: Explored various fields and identified potential project
  directions.
* First-Stage Experimental Training: Gained hands-on experience with fundamental lab techniques and
  safety rules.

**2. Human Practices:**

* Plasmid Campus Orienting Activity: Combined science outreach with outdoor engagement.
* Continued operating our WeChat official account to record activities and collect feedback.

**3. Model:**

* Learned and practiced basic modeling methods to support project design.

**4. Art:**

* Designed our team flag and logo to showcase creativity and identity.

**5. Wiki:**

* Started training on wiki editing and content organization.

![img](https://static.igem.wiki/teams/5855/notebook/april.webp)

## May

**1. Wet Lab:**

* Brainstorming & Literature Review: Explored different research fields and read relevant papers.
* Second-Stage Experimental Training.
* Learned a series of experimental operations with *E. coli*.
* Reinforced laboratory safety awareness.
* Continued reading papers related to synthetic biology and our project.

**2. Human Practices:**

* Hold the Gene Island Exploration Program with Qingjiang High School's students.
* Carried out the 2025 National Science and Technology Week Popularization Activity.
* Continued operating our WeChat official account to record activities and collect feedback.
* Proposed the "Double-Helix Model" for this year's IHP section and the ATGC framework for the
  Education section.

**3. Model:**

* Continued learning modeling methods to support project design.

**4. Art:**

* Designed creative memes and team merchandise.

**5. Wiki:**

* Learned wiki editing and webpage construction.

![img](https://static.igem.wiki/teams/5855/notebook/may.webp)

## June

**1. Wet Lab:**

* Determined the project: Temperature-controlled one-step synthesis of colored bacterial cellulose.
* Determine *E. coli* as engineered bacteria.
* Built the gene circuit, read papers related to our project.

**2. Human Practices:**

* Discussed with our PI, Professor Chen Xi, determining our project.
* Consulted with Professor Zhang Chong, optimizing our experiment's design.
* Planned Human Practices activities.

![img](https://static.igem.wiki/teams/5855/notebook/june-zhongchong.webp)

**3. Art:**

* Designed diagrams for the wet lab pathway.
* Designed diagrams for the application of our project.
* Designed team visuals.

**4. Model:**

* Explored and studied the project's concepts.
* Reviewed relevant literature.
* Developed modeling approaches.

**5. Wiki:**

* Prepared wiki content to fully support the project.

## July

**1. Wet Lab:**

* Optimized the design of the gene circuit.
* Verified the function of the gene circuit.
* Consulted relevant experts to address experimental challenges.

![img](https://static.igem.wiki/teams/5855/notebook/july2.webp)

**2. Human Practices:**

* Launched “Little Fabric Artist in Leaf Dyeing Workshop” Popular Science Activity.
* Collaborated with NNU-CHINA, engaging in team exchanges and knowledge sharing.
* Visited Nanjing Institute of Product Quality Inspection, gaining insights into textile testing
  standards and practices.
* Interviewed with Prof. Hua Shen, professor at Donghua University, expert in textile materials.

![img](https://static.igem.wiki/teams/5855/notebook/july.webp)

**3. Model:**

* Conducted molecular and mathematical modeling closely aligned with the project, providing support
  for experimental design and analysis.

**4. Art:**

* Designed our project poster.
* Built the outline of our promotion video.

![img](https://static.igem.wiki/teams/5855/notebook/july-poster.webp)

**5. Wiki:**

* Learned wiki editing and webpage construction to prepare for our ICII web platform.

## August

**1. Collaborations & Conferences:**

1. Attended the 12th CCiC.

2. Communicated with
HKU-HongKong both face-to-face and online, followed by completing the **E. coli** handbook.

3. Held an online meeting with UM-Macau.

![img](https://static.igem.wiki/teams/5855/notebook/august.webp)

**2. Wet Lab:**

* Optimized the expression of bacterial cellulose.
* Completed experiments related to TyrBm.
* Complete the experients on contribution.

**3. Human Practices:**

* Visited to Qingdao Textile Museum History Pavilion, exploring the dialogue between traditional
  and modern textile industries.
* Visited the Intertextile SHANGHAI Home Textiles Fair, gaining insights into trends and
  sustainable fabrics.
* Launched "Passing Love with Old Clothes, Popularizing Science with Us" activities, promoting
  recycling and science outreach.
* Joined SynCamp Unconference to clarify the industrialization pathway for bacterial cellulose
  fabrics.
* Designed the framework of our ICII webpage.
* Online thematic articles:

  + The Use of Synthetic Biology in Clothing.
  + The Use of Synthetic Biology in Food.

![img](https://static.igem.wiki/teams/5855/notebook/august2.webp)

**4. Model:**

* Conducted both molecular modeling and mathematical modeling closely aligned with the project.

**5. Art:**

* Released the NAU-CHINA 2025 Promotion Video.
* Designed and produced team merchandise.

**6. Wiki:**

* Started building the ICII webpage.

## September

**1. Wet Lab:**

* Verified the overall gene circuit and results.
* Completed the improvement part.
* Submitted the safety form.

**2. Human Practices:**

* Online thematic articles:

  + The Use of Synthetic Biology in Housing: showcased novel applications of synthetic
    biology in
    housing.
  + The Use of Synthetic Biology in Transportation: explored innovative ways synthetic
    biology may
    change transportation.
* Launched Campus Promotion Activity for Bacterial Cellulose Project, promoting synthetic biology
  through campus outreach.
* Launched "Modern Silk Road" Themed Monopoly Game.

![img](https://static.igem.wiki/teams/5855/notebook/september.webp)

**3. Model:**

* Completed basic solving of the models.
* Proposed and developed a fashion-related model.

**4. Art&Wiki:**

* Designed creative works and started wiki construction.

## October

**1. Wet Lab:**

* Completed experiments.

**2. Human Practices:**

* Consulted with Jen Keane, Co-founder and CEO of Modern Synthesis.

![img](https://static.igem.wiki/teams/5855/notebook/oct.webp)

**3. Model:**

* Completed models.

**4. Art:**

* Polished wiki.

**5. Wiki:**

* Finished wiki edit.

Note: All photos released in this page have been approved by the individuals involved.

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Engineering
 Success

"Design, build, test and learn, from careful steps to bold strides; success is engineered, not
accidental."
— NAU-CHINA

![Classroom Scene](https://static.igem.wiki/teams/5855/heroimg/engineering.webp)

Cellulose Synthesis Module

Loop 1

Loop 2

Dyeing module

Loop 1

Loop 2

Loop 3

Loop 4

Loop 5

Temperature Regulation Module

Loop1

Loop2

Waterproof Module

Loop 1

Loop 2

Loop 3

References

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![指示器](https://static.igem.wiki/teams/5855/generalimg/mvsz-half.webp)

## Cellulose Synthesis Module

### Loop 1

#### Design

The *bcs* operon of cellulose-producing *Komagataeibacter sucrofermentans* species comprises
four structural genes, *bcs*A, *bcs*B, *bcs*C and *bcs*D, that act in concert to
assemble and export BC (Figure 1). Among them, *bcs*A encodes a membrane-associated
glycosyltransferase that, once activated by the second messenger c-di-GMP, transfers glucose from
UDP-glucose to a growing β-1,4-glucan chain. Its periplasmic partner BcsB binds c-di-GMP, stabilizes
BcsA, and directs the nascent polymer across the inner membrane; the BcsAB complex is therefore the
essential catalytic core. BcsC forms an outer-membrane channel for polymer extrusion, while BcsD
promotes periplasmic crystallization into ordered microfibrils.

![Figure 1. Genetic organization of <i>bcs</i> operon<sup>[1]</sup>.](https://static.igem.wiki/teams/5855/engineering/figure-1.webp)

Figure 1. Genetic organization of *bcs* operon[1].
 When BcsA (green)
is activated by
c-di-GMP, it incorporates glucose units into a cellulose chain in the cytoplasm using UDP glucose as a
substrate. BcsB (blue) guides the glucan chain through the periplasm; BcsD (orange) crystallizes four
glucan chains in the periplasm, and finally, BcsC (gray) exports the BC micro-fibrils into the
extracellular space.

Through literature review[2], we learned that there are differences
between *E. coli* DH5α and BL21(DE3) when used as host strains for expressing *bcs*ABCD. The
selection of host strains is crucial. Since the "one-step" system is designed for bacterial
cellulose (BC) synthesis at 25°C, we first investigated which strain-*E. coli* DH5α or BL21(DE3)-is
more suitable for BC expression at this temperature.

#### Build

Given the excessively long length of the full, *bcs*ABCD gene cluster (9,126 bp), we divided these
four core genes into two segments, namely *bcs*AB and *bcs*CD, to facilitate their expression
in *E. coli*. Specifically, the *bcs*A and *bcs*B genes were assembled into the
pACYCDuet-1 vector to construct the plasmid pACYCDuet-1-bcsAB (Figure 2a), while *bcs*C
and *bcs*D were inserted into the pQE-60 vector to construct pQE-60-bcsCD (Figure 2b).
However, the original pACYCDuet-1 vector harbors a T7 promoter-a regulatory element incapable of driving
gene expression in *E. coli* DH5α, as this strain lacks T7 RNA polymerase. To address this
limitation, we first cloned *bcs*AB into the pQE-60 vector to construct an intermediate plasmid.
Subsequently, we cloned the *bcs*AB fragment (along with the T5 promoter and lambda t0 terminator
derived from pQE-60) into the pACYCDuet-1 vector to complete the construction of pACYCDuet-1-bcsAB. The
*bcs*AB gene fragment (Figure 2c), *bcs*CD gene fragment (Figure 2c), pQE-60 vector fragment
(Figure 2d), T5 promoter-*bcs*AB-lambda t0 terminator gene fragment (Figure 2e) and pACYCDuet-1
vector fragment (Figure 2e) were amplified by PCR, and the resulting components were subjected to
In-fusion Cloning.

![Figure 2. Plasmid map, PCR verification and sequencing identification of recombinant plasmids                 pACYCDuet-1-bcsAB and pQE-60-bcsCD. a. Plasmid map of pACYCDuet-1-bcsAB. b. Plasmid map of pQE-60-bcsCD.](https://static.igem.wiki/teams/5855/engineering/figure-2.webp)

Figure 2. Plasmid map, PCR verification and sequencing identification of recombinant
plasmids
pACYCDuet-1-bcsAB and pQE-60-bcsCD.
a. Plasmid map of pACYCDuet-1-bcsAB. b. Plasmid map of
pQE-60-bcsCD. c. 1% agarose gel electrophoresis of PCR-amplified bcsAB (4,675 bp) and bcsCD (4,451 bp).
M: DNA marker;
Lane 1: bcsAB; Lane 2: bcsCD. d. 1% agarose gel electrophoresis of PCR-amplified pQE-60 vector (3,423
bp). M: DNA marker. e. 1% agarose gel electrophoresis of PCR-amplified T5 promoter-bcsAB-lambda
t0-terminator (4,915 bp) and pACYCDuet-1 vector (2,276 bp). M: DNA marker; Lane 1: T5
promoter-bcsAB-lambda t0-terminator; Lane 2: pACYCDuet-1 vector. f. Sequencing results of the
recombinant plasmid pACYCDuet-1-bcsAB. g. Sequencing results of the recombinant plasmid pQE-60-bcsCD.

Next, the recombinant plasmids were transformed into *E. coli* DH5α. Based on the sequencing
results (Figure 2f and 2g), we successfully obtained the plasmids pACYCDuet-1-bcsAB and pQE-60-bcsCD.
These two recombinant plasmids were then co-transformed into *E. coli* DH5α and BL21(DE3) strains,
respectively.

#### Test

Both engineered strains were cultured and induced for expression in LB medium. When the OD600
value reached 0.8, IPTG was added to a final concentration of 0.05 mmol/L to induce expression under the
conditions of 25°C and 180 rpm, with a control group set up simultaneously. After 6 h, visible gel-like
dispersed structures were observed in the medium containing the DH5α strain (Figure 3a), while no such
phenomenon was detected in the medium with the BL21(DE3) strain. After another 12 h, 1 mL of induced
cells was harvested via centrifugation at 10,000 × *g* for 10 min at 4°C. The harvested cells were
first washed twice with nuclease-free water, then mixed with 2× sample buffer, and finally boiled for 5
min to denature the proteins. These processed samples were subsequently used for SDS-PAGE and Coomassie
Brilliant Blue staining analysis (Figure 3b).

![Figure 3. Expression of recombinant BcsABCD.](https://static.igem.wiki/teams/5855/engineering/figure-3.webp)

Figure 3. Expression of BcsABCD
 a. BC was detected as white gel-like
dispersed structures in
DH5α 6 h after 0.05 mM IPTG induction. b. SDS-PAGE of expression products of BcsABCD. Lane 1: Protein
ladder; Lane 2-3: DH5α protein samples obtained from induction with 0.05 mmol/L IPTG and from the
uninduced group, respectively. Lane 4-5: BL21 protein samples obtained from induction with 0.05 mmol/L
IPTG and from the uninduced group, respectively.

The results showed that all four key proteins were
detected as the expected molecular weights in both DH5α and BL21 strains. However, visible BC was only
formed in DH5α. In contrast, although BL21 (DE3) also expressed the *bcs*ABCD operon under the same
conditions, it failed to produce visible BC.

#### Learn

Based on the result above, we reviewed relevant literature and identified the potential reasons for the
differences in BC expression between the DH5α and BL21(DE3): first, the membrane-associated BcsABCD
required for BC synthesis exhibit toxicity when overexpressed in BL21(DE3), disrupting critical
physiological functions of the host cell membrane and interfering with the normal cellular environment
necessary for BC synthesis; second, although SDS-PAGE analysis showed that BL21(DE3) could express
recombinant *bcs* family proteins after induction, these proteins may form inactive inclusion
bodies due to abnormal folding, preventing their assembly into functional BC synthesis complexes with
"catalysis-transport" capabilities[2]. Therefore, we selected DH5α for subsequent BcsABCD
expression experiments.

### Loop 2

#### Design

The overexpression of the BcsABCD requires careful balancing. Insufficient induction leads to low-level
BC production, whereas excessive induction risks protein misfolding, inclusion body formation, and
metabolic burden. To address this, we needed to determine the optimal induction conditions for the
BcsABCD in DH5α.

#### Build

To determine the optimal IPTG concentration for inducing the expression of the BcsABCD, we designed the
following concentration gradient: 0, 0.05, 0.1, 0.5, and 1.0 mmol/L. The expression conditions were the
same as before (25°C, 180 rpm, 18 h).

#### Test

After 6 h, gel-like dispersed structures were first observed in the medium with a final IPTG
concentration of 0.05 mmol/L, followed by those in the media containing 0, 0.1, 0.5 mM IPTG. After
another 12 h, denatured proteins were obtained following the previous methods for SDS-PAGE and Coomassie
Brilliant Blue staining analysis.

![Figure 4. SDS-PAGE of expression products of BcsABCD under different induction conditions in DH5α.](https://static.igem.wiki/teams/5855/engineering/figure-4.webp)

Figure 4. SDS-PAGE of expression products of BcsABCD under different induction conditions
in DH5α.
Lane
1: Protein ladder; Lane 2-6: DH5α protein samples derived from induction with 0, 0.05, 0.1, 0.5 and 1.0
mmol/L IPTG, respectively.

It could be observed that the expression levels of BcsABCD were relatively
higher in the groups induced with 0, 0.05, and 0.1 mM IPTG, but the overall difference among all groups
was not significant (Figure 4). Given that *bcs*A catalyzes BC synthesis using UDP-glucose as a
direct substrate, DH5α first converts glucose in the medium into UDP-glucose. Therefore, changes in the
glucose concentration in the culture medium should be correlated with BC production. To further evaluate
the optimal induction conditions, we quantified the temporal changes in glucose concentration in the
culture medium during the cultivation of the DH5α strain. We sampled at 0, 6, 18, and 24 h respectively,
determined the glucose concentration in the medium, and plotted a glucose concentration curve based on
these data (Figure 5), aiming to indirectly calculate the BC synthesis rate.

![img](https://static.igem.wiki/teams/5855/engineering/23456-1.webp)

Figure 5. Changes in glucose concentration during cultivation of DH5α strain under
induction with
different IPTG concentrations.
a. Schematic diagram of glucose concentration changes in 96-well
plates
at different incubation times and IPTG concentrations. b. Time-dependent curve of glucose concentration
in medium during cultivation of DH5α strain induced with different IPTG concentrations

Notably, for the
groups with 0 mmol/L and 0.05 mmol/L IPTG, the glucose concentration first increased and then decreased
over time, and the glucose in the medium barely changed after approximately 18 h. We used the following
formulas to determine the synthesis rate and consumption rate:
$$\text{Glucose synthesis rate
(mg/(L}\cdot\text{h))} = \frac{\text{Glucose concentration|}\_\text{0 h}^\text{6 h}
}{\text{Time interval|}\_\text{0 h}^\text{6 h} }$$
$$\text{Glucose consumption rate (mg/(L}\cdot\text{h))} = \frac{\text{Glucose concentration|}\_\text{6
h}^\text{18 h}
}{\text{Time interval|}\_\text{6 h}^\text{18 h} }$$ The results showed that the group induced with 0.05
mmol/L IPTG
exhibited the highest glucose utilization efficiency, with a synthesis rate of 28.7 mg/(L·h) and a
consumption rate of 13.7 mg/(L·h). This was followed by the 0 mmol/L IPTG group, where the synthesis
rate and consumption rate were 11.6 mg/(L·h) and 6.43 mg/(L·h) respectively, which also indicates that
the leaky expression of the T5 promoter is relatively severe. In contrast, the groups induced with
higher IPTG concentrations (≥0.1 mmol/L) showed lower and relatively similar glucose utilization
efficiencies, with almost no change in glucose concentration in their culture media.

#### Learn

At an IPTG induction concentration of 0.05 mmol/L, the engineered bacteria balanced the expression of
BcsABCD and the host metabolic burden, thereby achieving the highest glucose utilization efficiency to
support efficient BC production. In contrast, higher IPTG concentrations might cause metabolic
disturbance, making it difficult for the engineered bacteria to synthesize BC.

## Dyeing module

### Loop 1

#### Design

Bacterial cellulose (BC) is naturally colorless and lacks visual diversity, which limits its direct
application in textiles and functional materials. To overcome this, we aimed to endow BC with stable
coloration by introducing natural pigments. We chose eumelanin, a biopolymer pigment with high stability
and biocompatibility. Its synthesis requires the oxidation of L-tyrosine, and tyrosinase (Tyr) is the
key catalyst for this step (Figure 6)[3]. We selected tyrosinase (TyrBm) derived from *Bacillus
megaterium* as the catalyst[4]. This enzyme has been proven to achieve efficient
expression in *E. coli* BL21(DE3) cultured in TB medium and catalyze the oxidation reaction of
L-tyrosine. Since the subsequent "one-step" system needs to be implemented in LB medium, we first
verified whether TyrBm could achieve soluble expression in LB medium.

![img](https://static.igem.wiki/teams/5855/engineering/figure-6.webp)

Figure 6. Eumelanin biosynthetic pathway.

#### Build

We constructed plasmid pET-28a(+)-TyrBm (Figure 7a). The *TyrBm* gene fragment (Figure 7b) and
pET-28a(+) vector fragment (Figure 7c) were amplified by PCR, and the resulting components were
subjected to In-fusion Cloning.

![img](https://static.igem.wiki/teams/5855/engineering/figure-7.webp)

Figure 7. Plasmid map of pET-28a(+)-TyrBm and 1% agarose gel electrophoresis of
PCR-amplified pET-28a(+)-TyrBm components.
a. Plasmid map of pET-28a(+)-TyrBm. b. 1% agarose gel
electrophoresis of PCR-amplified pET-28a(+) vector (5,326 bp). M: DNA marker. c. 1% agarose gel
electrophoresis of PCR-amplified TyrBm (909 bp). M: DNA marker.

Next, the recombinant plasmid was transformed into *E. coli* DH5α. Based on the sequencing results
(Figure 8), we successfully obtained the plasmid pET-28a(+)-TyrBm. Then, the plasmid was transformed
into *E. coli* BL21(DE3).

![img](https://static.igem.wiki/teams/5855/engineering/figure-8.webp)

Figure 8. Sequencing results of the recombinant plasmid pET-28a(+)-TyrBm.

#### Test

We expressed TyrBm in LB and TB media respectively. When the OD600 reached 0.6, the expression
was induced by IPTG under the conditions of 37°C and 150 rpm. After 12 h, the color changes of the
cultures were observed (Figure 9a). After harvesting the cell pellet, it was resuspended in binding
buffer (20 mmol/L sodium phosphate buffer, pH 7.5, 500 mmol/L NaCl; and 20 mmol/L imidazole), ultrasonic
cell disruption (power: 50 W, work time: 1.0 s, interval: 2.5 s, total time: 40 min, ice bath) was
performed to separate the supernatant and precipitate (16,000 × *g*, 15°C, 20 min), followed by
SDS-PAGE and Coomassie Brilliant Blue staining analysis (Figure 9b). The results showed that TyrBm
(39.26 kDa) could be expressed in both LB and TB media. However, there were two problems: first, in both
media, the expressed TyrBm was mainly distributed in the precipitate with low soluble expression level;
second, the TB medium turned significantly black after induction, while no obvious blackening was
observed in LB medium.

![img](https://static.igem.wiki/teams/5855/engineering/figure-9.webp)

Figure 9. Expression of recombinant TyrBm under TB/LB culture conditions.
 a. Colors of
each group of cultures after 12 h of TyrBm expression. -: No addition of IPTG; +: Addition of IPTG. b.
SDS-PAGE of expression products of TyrBm. Lane 1: Protein ladder; Lane 2-4: Whole-cell lysate,
supernatant and pellet from uninduced cells in TB medium, respectively; Lane 5-7: Whole-cell lysate,
supernatant and pellet from uninduced cells in LB medium, respectively; Lane 8-10: Whole-cell lysate,
supernatant and pellet from IPTG induced cells in TB medium, respectively; Lane 11-13: Whole-cell
lysate, supernatant and pellet from IPTG induced cells in LB medium, respectively.

#### Learn

The above experiments confirmed that LB medium could be used as the subsequent optimization medium.
Regarding the issue of low soluble expression levels, we hypothesized that induction intensity might be
an influencing factor. For the problem that TyrBm was expressed in LB medium but no blackening was
observed, we compared the components of TB and LB media and proposed that the blackening phenomenon in
TB medium might be attributed to its higher content of tryptone and yeast extract (which are rich in
amino acids and trace metal ions). Therefore, we hypothesized that the presence of substrates/cofactors
(L-tyrosine and Cu2+) in the medium might affect the formation of blackened products.

### Loop 2

#### Design

Literature reports indicate that the substrate for the tyrosinase-catalyzed reaction is L-tyrosine, and
the activity/assembly of the enzyme depends on Cu2+; common systems are supplemented with 0.5
g/L L-tyrosine and 10 μmol/L CuSO4. To investigate the effects of substrates/cofactors on the
soluble expression of TyrBm and product formation, we designed an IPTG gradient experiment and set up
controls with or without substrates and cofactors in the medium.

#### Build

Six experimental combinations were designed (Table 1), including three levels of IPTG (0, 0.5, 1 mmol/L)
and controls with or without substrates and cofactors (0.5 g/L L-tyrosine + 10 μmol/L CuSO4)
in the medium.

Table 1. Detailed information of experimental combinations

| Number | IPTG（mmol/L） | Substrates and cofactors addition (0.5 g/L L-tyrosine + 10 μmol/L CuSO4) |
| --- | --- | --- |
| 1 | 0 | - |
| 2 | 0 | + |
| 3 | 0.5 | - |
| 4 | 0.5 | + |
| 5 | 1 | - |
| 6 | 1 | + |

-: No addition of 0.5 g/L L-tyrosine + 10 μmol/L CuSO4; +: Addition of 0.5 g/L
L-tyrosine + 10 μmol/L CuSO4.

The colors of each group before expression were shown in Figure 10a. The expression conditions were the
same as before (37°C, 150 rpm, 12 h).

#### Test

The color changes of the cultures were observed (Figure 10a), and each group of samples was subjected to
ultrasonic cell disruption, supernatant-precipitate separation, SDS-PAGE, and Coomassie brilliant blue
staining analysis (Figure 10b).

![img](https://static.igem.wiki/teams/5855/engineering/figure-10.webp)

Figure 10. Expression of recombinant TyrBm among the 6 experimental combinations.
a.
Color changes of cultures in each group (Table 1) after 12 h of TyrBm expression. b. SDS-PAGE of
expression products of TyrBm. Lane 1: Protein ladder; Lanes 2–3: Supernatant and pellet from uninduced
cells in LB medium without substrates and cofactors, respectively; Lanes 4–5: Supernatant and pellet
from uninduced cells in LB medium with substrates and cofactors, respectively; Lanes 6–7: Supernatant
and pellet from 0.5 mmol/L IPTG induced cells in LB medium without substrates and cofactors,
respectively; Lanes 8–9: Supernatant and pellet from 0.5 mmol/L IPTG induced cells in LB medium with
substrates and cofactors, respectively; Lanes 10–11: Supernatant and pellet from 1 mmol/L IPTG induced
cells in LB medium without substrates and cofactors, respectively; Lanes 12–13: Supernatant and pellet
from 1 mmol/L IPTG induced cells in LB medium with substrates and cofactors, respectively.

The results showed that the soluble expression level of TyrBm was relatively high under two conditions:
0.5 mmol/L IPTG with the addition of L-tyrosine and Cu2+, and 1 mmol/L IPTG without the
addition of L-tyrosine and Cu2+ (Figure 5c). Given that our "one-step" system requires the
addition of substrates and cofactors at the beginning of the dyeing module to accelerate the dyeing
process, we selected 0.5 mmol/L IPTG as the appropriate induction concentration. Notably, under the
condition of LB with substrates and cofactors, the culture medium showed obvious blackening even without
IPTG addition (Figure 5b). This suggested that in LB medium, tyrosine oxidation/polymerization or
spontaneous discoloration triggered by medium components occurs even without induction.

#### Learn

We confirmed that 0.5 mmol/L IPTG was the appropriate induction concentration. However, non-induced
blackening occurred when substrates and cofactors were added to LB medium. We hypothesized that
components in the medium might be involved in the spontaneous oxidation of L-tyrosine.

### Loop 3

#### Design

It was necessary to clarify the cause of blackening of TyrBm under the condition of LB with substrates
and cofactors. We knew from previous studies that TB medium was commonly used as the conventional
expression system for TyrBm. Therefore, we first needed to confirm whether the same blackening
phenomenon occurred under the classic TB condition, so as to determine whether this phenomenon was
specific to the target protein or caused by the LB medium.

#### Build

Eight experimental combinations were designed (Table 2): LB or TB medium with ±IPTG (0 or 0.5 mmol/L) and
±substrates and cofactors (0.5 g/L L-tyrosine + 10 μmol/L CuSO4).

Table 2. Detailed information of experimental combinations.

| Number | Medium type | IPTG (mmol/L) | Substrates and cofactors addition (0.5 g/L L-tyrosine + 10 μmol/L CuSO4) |
| --- | --- | --- | --- |
| 1 | LB | 0 | - |
| 2 | LB | 0 | + |
| 3 | LB | 0.5 | - |
| 4 | LB | 0.5 | + |
| 5 | TB | 0 | - |
| 6 | TB | 0 | + |
| 7 | TB | 0.5 | - |
| 8 | TB | 0.5 | + |

-: No addition of 0.5 g/L L-tyrosine + 10 μmol/L CuSO4; +: Addition of 0.5 g/L
L-tyrosine + 10 μmol/L CuSO4.

The expression conditions were the same as before (37°C, 150 rpm, 12 h).

#### Test

The colors of the cultures were observed (Figure 11).

![img](https://static.igem.wiki/teams/5855/engineering/figure-11.webp)

Figure 11. Colors of each group (Table 2) of cultures after 12 h of TyrBm expression.

The results showed that under the non-induced condition (0 mmol/L IPTG) with substrates and cofactors
addition, the LB culture medium turned significantly black, while no blackening was observed in TB
culture.

#### Learn

The experiment indicated that the difference in composition between the two media might be a key factor
contributing to the non-induced blackening of substrates in LB medium.

### Loop4

#### Design

The main difference between TB medium and LB medium lies in that TB medium contains an additional 0.4%
(v/v) glycerol and 89 mmol/L sodium phosphate buffer. We hypothesized that either one of these two
components or their synergistic effect inhibited the non-induced oxidation of L-tyrosine, thereby
reducing blackening.

#### Build

Using LB medium as the base, we employed the controlled variable method to add 0.4% (v/v) glycerol or 89
mmol/L sodium phosphate buffer. Against a background containing substrates and cofactors, we set up six
experimental groups (Table 3) with IPTG concentrations of 0 or 0.5 mmol/L.

Table 3. Detailed information of experimental combinations.

| Number | Medium type | IPTG (mmol/L) | 89 mmol/L sodium phosphate buffer | 0.4% (v/v) glycerol | Substrates and cofactors addition (0.5 g/L L-tyrosine + 10 μmol/L CuSOCuSO4) |
| --- | --- | --- | --- | --- | --- |
| 1 | LB | 0.5 | - | - | + |
| 2 | LB | 0 | - | - | + |
| 3 | LB | 0.5 | - | + | + |
| 4 | LB | 0 | - | + | + |
| 5 | LB | 0.5 | + | - | + |
| 6 | LB | 0 | + | - | + |

-: No addition of the corresponding component; +: Addition of the corresponding
component.

The expression conditions were the same as before (37°C, 150 rpm, 12 h).

#### Test

The colors changes of the media of the were observed (Figure 12).

![img](https://static.igem.wiki/teams/5855/engineering/figure-12.webp)

Figure 12. Culture supernatant obtained without bacteria after induction for 12 h of each group (Table 3).

The results showed that after adding glycerol or sodium phosphate buffer to the LB medium, the
non-induced blackening of the LB medium was significantly reduced, indicating that both could inhibit
this phenomenon to a certain extent. Among the two, the addition of glycerol exhibited the best
effect.

#### Learn

Based on the above results, we found that adding glycerol to the medium could inhibit the non-enzymatic
darkening phenomenon in LB medium. For the subsequent large-scale expression of TyrBm, we will adopt LB
supplemented with 0.4% (v/v) glycerol, 0.5 mmol/L IPTG, 0.5 g/L L-tyrosine, and 10 μmol/L
CuSO4 as the standard conditions.

### Loop5

#### Design

After completing the expression, we needed to determine the catalytic activity and kinetic
characteristics of TyrBm, clarify its monophenol oxidation activity (L-tyrosine -> L-DOPA) and diphenol
oxidation activity (L-DOPA -> dopaquinone), and obtain its kinetic parameters.

#### Build

Under the optimized culture conditions, TyrBm was induced for expression at 37°C and 150 rpm for 12 h,
with the group without substrates and cofactors addition used as a control. After expression, the cells
were disrupted by ultrasonication, and the supernatant and precipitate were separated. Subsequently,
Immobilized Metal Affinity Chromatography (IMAC) purification was performed using a HypurT Ni-NTA 6FF
(His-Tag) prepacked Gravity column (1 mL), with elution carried out using 20 mmol/L sodium phosphate
buffer (pH 7.5) containing 500 mmol/L NaCl and either 300 mmol/L or 500 mmol/L imidazole. The purified
enzyme was subjected to dialysis treatment against 50 mmol/L sodium phosphate buffer (pH 6.5) with 0.01
mmol/L CuSO4 at 4°C for 24 h.

#### Test

According to SDS-PAGE analysis, TyrBm achieved partial soluble expression under both sets of conditions
(Figure 13a). After IMAC elution, the main target band appeared in the 500 mmol/L imidazole buffer
fraction (Figure 13b).

![img](https://static.igem.wiki/teams/5855/engineering/111111-1.avif)

Figure 13. SDS-PAGE analysis of TyrBm expression products and IMAC-purified fractions.
a. SDS-PAGE of expression products of TyrBm. Lane 1: Protein ladder; Lanes 2-3: Supernatant and pellet from 0.5 mmol/L IPTG induced cells in LB medium with 0.5 g/L L-tyrosine and 10 µmol/L CuSO4, respectively; Lanes 4-5: Supernatant and pellet from 0.5 mmol/L IPTG induced cells in LB medium, respectively. b. SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column. Lane 1: Protein ladder; Lane 2-5: TyrBm supernate obtained from LB medium supplemented with with 0.5 g/L L-tyrosine and 10 µmol/L CuSO4 after being bound to Ni-NTA resin, eluted with 20, 300 and 500 mmol/L imidazole, respectively ; Lane 6-9: TyrBm supernate obtained from LB medium after being bound to Ni-NTA resin, TyrBm supernate eluted with 20, 300 and 500 mmol/L imidazole, respectively.

In a 96-well plate, L-tyrosine and L-DOPA were used as substrates to determine the monophenolase and
diphenolase activities of TyrBm, respectively. Besides, an increasingly obvious colour occurred in
experimental group compared to control group along with the increase of concentration (Figure 14). The
reaction system was incubated at 37°C, and the change in absorbance at 475 nm was recorded
(ε475=3,600/(mol·cm)). The amount of dopachrome produced was calculated using the formula ΔA
= A1 − A0, and the enzyme activity unit was converted based on this (1 U is
defined as the amount of enzyme required to produce 1 μmol of dopachrome per minute). Subsequently, the
specific activity was calculated according to the protein concentration (determined by the Bicinchoninic
Acid Assay method).

![img](https://static.igem.wiki/teams/5855/engineering/figure-14.webp)

Figure 14. The 96 Well Cell Culture Plates of tyrosinase TyrBm used for activity＆kinetic
assay and ΔA of each well.
 a. TyrBm purified from LB medium supplemented with L-tyrosine and
CuSO4. b. TyrBm purified from LB medium. Reaction system incubated at 37°C for 30 min,
absorbance measured at 475 nm (ε475=3,600/(mol·cm))

The results showed that the monophenolase activity without IPTG addition was 1,461 U/mg and the
diphenolase activity was 4,032 U/mg; with IPTG addition, the monophenolase activity was 35,648 U/mg and
the diphenolase activity was 199,346 U/mg. These results indicated that substrates and cofactors
significantly enhanced the catalytic activity of TyrBm. Subsequently, based on the group with the
highest specific activity, Michaelis-Menten and Lineweaver-Burk curves were plotted (Figure 15),
yielding the following Michaelis constant (Km) and maximum velocity (vmax)
results: for monophenol oxidation, Km = 34.37 μmol/L and vmax = 2.487
μmol/(L·min); for diphenol oxidation, Km = 540.84 μmol/L and vmax = 2.274
μmol/(L·min).

![img](https://static.igem.wiki/teams/5855/engineering/figure-15.webp)

Figure 15. The kinetic assay results of TyrBm.
 a-b. Michaelis-Menten plot of
enzymatic reaction from tyrosine to dopachrome experiments and from L-DOPA to dopachrome. c-d.
Lineweaver-Burk double reciprocal plot of enzymatic reaction from L-tyrosine to dopachrome experiments
and from L-DOPA to dopachrome.

**Dyeing Time Simulation via PDE Model**
 To quantitatively determine the specific dyeing duration,
a combined approach of mathematical modeling and biological simulation was conducted. In the
mathematical simulation, a partial differential equation (PDE) model was established to describe the
transport and diffusion of pigment molecules within the cellulose matrix. Using the finite element
method (FEV) for numerical computation, we obtained the temporal and spatial evolution of pigment
concentration across different regions. The simulation results revealed that the dyeing rate varied
significantly among local zones, which can be attributed to the porous and heterogeneous nature of the
cellulose structure. Due to the presence of pores with varying diameters, pigment penetration occurred
at different rates, leading to non-uniform color development across the material (Figure 16):

![img](https://static.igem.wiki/teams/5855/model/mathematic-model/pde/pde-result.webp)

Figure 16. Simulation slicing of the staining time calculation process using the PDE
algorithm.

Based on the simulation outcomes, the effective dyeing duration was determined to be approximately 37,825 s (≈10.5 h), corresponding to the point at which the pigment concentration within the cellulose
matrix reached equilibrium (Figure 17). Beyond this time, the diffusion flux approached zero, and the
color intensity exhibited minimal further change. This result provided a quantitative reference for
optimizing dyeing parameters in experimental and industrial applications, ensuring sufficient coloration
while avoiding unnecessary processing time.

![img](https://static.igem.wiki/teams/5855/engineering/20251008-214242.webp)

Figure 17. Computational visualization of the PDE algorithm for the quantitative analysis
of staining time dynamics.

If you are interested to learn more, please visit the Model page.

[The Model page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27mathematical-modeling%27%29%20%7D%7D)

#### Learn

It could be concluded from the experimental results that the supplementation of L-tyrosine and
Cu2+ significantly promoted the activity and stability of TyrBm. TyrBm exhibited a high
affinity for L-tyrosine, while the oxidation rate of L-DOPA was slightly faster, which was consistent
with its natural catalytic order. A dual-pore PDE model was also built to characterize cellulose dyeing;
simulations showed most nodes reach dye equilibrium at approximately 37,825 s, some unsaturated.

## Temperature Regulation Module

### Loop1

#### Design

The temperature regulation module played a crucial role in our weaving and dyeing platform. It contained
three main parts: CI857, FourU riboswitch and PhlF repressor. To better demonstrate the module's
performance, we designed a testing circuit (Figure 18). Once induced by IPTG, CI857 expresses. Under
25°C, CI857 forms dimers that bind with R promoter to confine the combination of RNA polymerase with the
promoter, thus blocking the expression of PhlF and mRFP1. Meanwhile, FourU stem loop comes into being
inhibiting the RBS and again weakens the repressor and the fluorescence protein. The restriction of PhlF
initiates the translation of sfGFP guided by PhlF promoter that is inhibited by the repressor. Under
37°C, however, CI857 dimers disassociate, which eliminates the inhibition of R promoter while FourU
exposed the RBS binded with it, making ribosome possible to bind with RBS[5, 6]. As a result,
mRFP1 and PhlF emerge smoothly, the latter integrated with PhlF promoter, decreasing the synthesis of
sfGFP. In short, mRFP1 expresses more at higher temperature and sfGFP possesses stronger fluorescence
intensity at lower temperature.

![img](https://static.igem.wiki/teams/5855/engineering/figure-18.webp)

Figure 18. The testing circuit of the temperature regulation circuit that can express
proteins with
different fluorescence at distinct temperatures.

#### Build and Test

To ensure the feasibility of our temperature regulation module, mathematical modeling was processed to
predict the structure transformation of FourU. The simulation mainly focused on the binding of UUUU
sequence of the riboswitch with the SD sequence of RBS (AGGAGG). Firstly, we leveraged RNA Structure
developed by Mathews Laboratory to to forecast secondary structures of FourU at distinct temperatures
and discovered stable base pairing between AGGAGG and UUUU at 310 K (36.85°C), partially disrupted
pairing at 430 K (156.85°C) and completely disassembled pairing at 520 K (246.85°C) (Figure 19).

![img](https://static.igem.wiki/teams/5855/engineering/figure-19.webp)

Figure 19. Predicted secondary structures from RNA Structure of FourU at different
temperatures.

The
secondary structure prediction demonstrated the potential of FourU to regulate genes behind them, but
the precise transition temperature should only be forecasted with a quantitative analysis. Hence, a
translation initiation potential σ was introduced:
$$\sigma=\exp (-\frac{1}{RT}(E\_{total}))$$
To be
specific, R = 1.987 × 10−3 kcal/(mol⋅K) is the Boltzmann constant, T is the absolute
temperature, and Etotal could be calculated by the formula:
$$E\_{total}=E\_{SD}+E\_{tRNA}+E\_{stem}+E\_{4U{binding}}+E\_{coop}$$
Whereupon, we used Python to plot a normalized translation potential-temperature curve, finding out that
38°C was the unchaining temperature for the riboswitch, similar to the expected transition temperature
(37°C) (Figure 20). As for the subtle error, it was suspected that compared to the real circumstances,
our model could not cover all situation variates. For instance, the ionic environment was simplified to
sodium. More information about our modeling was provided at Model page.

[The Model page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27mathematical-modeling%27%29%20%7D%7D)

In a word, 37°C was a suitable transition
temperature for our project, which could be further authenticated with experiment.

![img](https://static.igem.wiki/teams/5855/engineering/figure-20.webp)

Figure 20. Temperature response curve of FourU. After the primary verification of FourU,
the plasmid
construction took place.

At first, we planned to use single fragment In-Fusion cloning to link the gene
fragment and the vector (Figure 21).

![img](https://static.igem.wiki/teams/5855/engineering/figure-21.webp)

Figure 21. The scheme of single fragment In-Fusion cloning for
pET-28a(+)-CI857-FourU-PhlF-mRFP1-sfGFP.
SfGFP was amplified from mRFP1-sfGFP (Figure 22).

![img](https://static.igem.wiki/teams/5855/engineering/figure-22.webp)

Figure 22. 1% agarose gel electrophoresis of the PCR amplified sfGFP (799 bp).

Whereafter, we amplified
the 7,587 bp pET-28a(+)-CI857-FourU-PhlF-mRFP1 vector by TCI-F (5'-GATCCGGCTGCTAACAAAGCC-3') and TCI-R
(5'-ACCTTAACGATACGGTACGTTTCGTAT-3') with the following procedure but failed (Table 4).

Table 4. PCR
procedure for linearized pET-28a(+)-CI857-FourU-PhlF-mRFP1 vector.

| Temperature(°C) | Time | Cycles |
| --- | --- | --- |
| 95 | 30 s | ×1 |
| 95 | 15 s | ×30 |
| 56 | 15 s | ×30 |
| 72 | 4 min | ×30 |
| 72 | 5 min | ×1 |

Hence, primers were inspected carefully whose base pair amount, annealing temperature and GC content were
all able to successfully amplify the target fragment. Given the correctness of primer design, we began
to think whether our procedure had made amplification difficult. Through discussion and searching for
alternative procedures from articles, specifications and the Internet, two optimized strategies were
carried out. For one, we adjusted the original extension time span to provide DNA polymerase with more
time to form the DNA strand complementary to template. Meanwhile, cycles were decreased from 30 to 28 so
as to reduce the opportunity of non-specific binding (Table 5).

Table 5. Optimized PCR procedure for
linearized pET-28a(+)-CI857-FourU-PhlF-mRFP1 vector.

| Temperature(°C) | Time | Cycles |
| --- | --- | --- |
| 95 | 30 s | ×1 |
| 95 | 15 s | ×28 |
| 56 | 15 s | ×28 |
| 72 | 8 min | ×28 |
| 72 | 5 min | ×1 |

For another, Touch Down two-step PCR approach was given a trial, which started at a relatively high
temperature, confining non-specific binding whereas at a lower temperature, correct products with a
higher concentration made the amplification more accurate (Table 6).

Table 6. Touch Down PCR procedure
for linearized pET-28a(+)-CI857-FourU-PhlF-mRFP1 vector.

| Temperature(°C) | Time | Cycles |
| --- | --- | --- |
| 95 | 3 min | ×1 |
| 95 | 15 s | ×5 |
| 74 | 8 min | ×5 |
| 95 | 15 s | ×5 |
| 72 | 8 min | ×5 |
| 95 | 15 s | ×5 |
| 70 | 8 min | ×5 |
| 95 | 15 s | ×25 |
| 68 | 8 min | ×25 |
| 68 | 5 min | ×1 |

Unfortunately, the two new approaches was unable to acquire the linearized vector. Therefore, we gave up
the plotted In-Fusion coloning strategy and chose to adopt a multi-fragment In-Fusion cloning way
(Figure 23).

![img](https://static.igem.wiki/teams/5855/engineering/figure-23.webp)

Figure 23. The scheme of multi-fragment In-Fusion cloning for
pET-28a(+)-CI857-FourU-PhlF-mRFP1-sfGFP.

Primers were designed to break the vector
into two parts of 3,298 and 4,310 bp respectively, making the amplification possible (Figure 24). With
the final catalysis of Exnase MultiS, the construction of pET-28a(+)-CI857-FourU-PhlF-mRFP1-sfGFP was
eventually accomplished.

![img](https://static.igem.wiki/teams/5855/engineering/figure-24.webp)

Figure 24. 1% agarose gel electrophoresis of the PCR amplified
pET-28a(+)-CI857-FourU-PhlF-mRFP1 fragment
1 (4,310 bp) and pET-28a(+)-CI857-FourU-PhlF-mRFP1 fragment 2 (3,298 bp).

We transformed the plasmid
into *E. coli* DH5α and ensured its correctness with Sanger sequencing (Figure 25).

![img](https://static.igem.wiki/teams/5855/engineering/figure-25.webp)

Figure 25. Sanger sequencing confirmed the correct assembly of the
CI857-FourU-PhlF-mRFP1-sfGFP
plasmid.

#### Learn

Long fragment PCR has long been an obstacle in gene amplification since primers are easier to miss-prime
during long extending time whilst GC-rich region will stop DNA polymerizing[7]. Thus, PCR
procedures should be designed carefully to meet the demand of distinct target products. For instance, a
relatively long extending time guided by the specification is beneficial for the binding of DNA
polymerase with single string sequence whereas the reduction in cycles enhances the fidelity during the
amplification. If all changes fail to obtain the target sequence, different methods are worth a shot.
Touch Down behaves better at the acquisition of complicated or long fragments and the shift from single
fragment In-Fusion to multi-fragment In-Fusion cloning strategy acts as a much easier way if the
construction of recombinant plasmid is demanded later. Additionally, optimization of codons themselves
at synthesis is able to relieve the challenges at the following PCR especially after the decrease in GC
content. In this condition, however, restriction endonuclease is also an option since we attempted to
acquire the whole linearized vector, meaning that it was of necessity to add cutting sites when
synthesising plasmids.

### Loop2

#### Design

Due to *E. coli* BL21(DE3)'s efficient protein expression capacity, it was an ideal choice for
relative fluorescence testing.

#### Build

Samples were collected periodically to measure OD600 as well as sfGFP and mRFP1 intensity
using a microplate reader. The mRFP1 intensity was determined by using an excitation wavelength of 584
nm and an emission wavelength of 666 nm while the sfGFP intensity was measured with an excitation
wavelength of 480 nm and an emission wavelength of 520 nm. Relative fluorescence intensity over OD600
was leveraged calculated using the following formula[8]:
$${(\frac{\text{FL}}{\text{OD}})}\_\text{corrected}=\frac{\text{FL}-\text{FL}\_\text{bg}}{\text{OD}-\text{OD}\_\text{bg}}$$

#### Test

The tested right plasmid was transformed into *E. coli* BL21(DE3) to express fluorescent protein.
Colonies were then inoculated into 5 mL of LB medium and incubated at 37°C with rotation at 250 rpm for
16 h so as to establish the seed cultures. Afterwards, we shifted 50 μL of the cultures into 5 mL of of
LB medium at 37°C, 250 rpm until their OD600 reached 0.9, where they were then induced by 1
mmol/L IPTG and the cultures were incubated at 25°C, 30°C and 37°C for 34 h. Fluorescence emitted by
sfGFP
and OD600 at each temperature were measured at 6, 9, 12 and 30 h postinoculation by the
microplate reader with three repetitions (Figure 26).

![img](https://static.igem.wiki/teams/5855/engineering/20251008-205339.webp)

Figure 26. Figure 26. Relative fluorescence intensity over OD600 at different temperatures.

The unstable growth at the few hours after the addition of IPTG might lead to the relatively high standard error. However, since the line tended to go smoother, the reliability of our detection was guaranteened. As was depicted, with time passing, the difference of sfGFP signal at different temperatures enlarged. Since the sfGFP was placed at the same place of the Bcs operon anticipated to produce bacterial cellulose at 25℃ where the relative fluorescence intensity mounted to the highest, the strongest expression of the operon came into being at the temperature. Meanwhile, our platform initiated the dyeing module at 37℃. Under such temperature, the sfGFP expressed weakly from which it could be inferred that the Bcs operon expressed less enzymes making TyrBm and CYP102A dye without disturbance.

#### Learn

As for the expression of fluorescence proteins, these proteins were of no differnce with normal proteins,
hence where they are placed attaching crucial significance to its translation. To be specific, in our
experiment design, behind the PhlF repressor lied mRFP1 regulated by the same RBS. Therefore, we failed
to achieve the correct expression level of it, which was possibly because the second gene behind the RBS
expressed relatively poorer than the first. Hence, the next time we plan to conduct verification with
the usage of fluorescence protein, it is reasonable to add an RBS before it to enhance its
expression.

## Waterproof Module

### Loop 1

#### Design

Hydrophobicity of our bacterial cellulose (BC) was achieved with the employment of biofilm-surface
layer protein A (BslA) from *Bacillus subtilis* with a double cellulose binding module (dCBM).
After a comprehensive review of corellated research literature, it is certificated that the recombinant
protein can bind effectively to the BC, thus forming a waterproof coat (Figure 27)[9].

![img](https://static.igem.wiki/teams/5855/engineering/11-b-1008-1658.avif)

Figure 27. Ideal interaction of BslA-CBMs with BC fibres[10].

#### Build

First, we amplified the *bslA-dcbm* gene and the linear pET-28a(+) vector by the usage of PCR. Then,
In-fusion cloning was leveraged to construct pET-28a(+)-BslA-dCBM recombinant plasmid that was
transferred into *E. coli* DH5α (Figure 28).

![img](https://static.igem.wiki/teams/5855/engineering/20251008-193839.avif)

Figure 28. The plasmid map of pET-28a(+)-BslA-dCBM and 1% agarose gel electrophoresis of
the PCR
amplified pET-28a(+)-BslA-dCBM parts.
a. The plasmid map of pET-28a(+)-BslA-dCBM. b. 1% agarose gel
electrophoresis of the PCR amplified BslA-dCBM (918 bp) and pET-28a(+) vector (5,323 bp).

After colony
PCR and Sanger sequencing, we ensured the correctness of the plasmid we built (Figure 29).

![img](https://static.igem.wiki/teams/5855/engineering/figure-29.webp)

Figure 29. Verification of recombinant plasmid pET-28a(+)-BslA-dCBM.
a. 1% agarose gel
electrophoresis of
colony PCR of using T7 and T7 ter primers. b. The result of sequencing the BslA-dCBM of the recombinant
plasmid.

Next, the plasmid was transformed into *E. coli* BL21(DE3) to express protein in LB medium
at 30°C for 16 h induced by 0, 0.2, 0.5 and 1 mmol/L IPTG.

#### Test

Fusion protein as BslA-dCBM, AlphaFold and GROMACS were leveraged to evaluate its stability. The
mathematical simulation started with the prediction of the structure of BslA and CBM respectively by
AlphaFold (Figure 30). BslA structure prediction yielded ipTM = pTM = 0.69, indicating high confidence. BslA adopts a β-barrel fold, supporting hydrophobic interactions consistent with its biological role. CBM structure prediction yielded ipTM = pTM = 0.7, indicating high confidence. CBM adopts a β-sandwich fold, typical of carbohydrate-binding modules, enabling cellulose recognition and adhesive function.

![img](https://static.igem.wiki/teams/5855/engineering/figure-30.webp)

Figure 30. BslA and CBM predicted result by AlphaFold.

AlphaFold was also used to forecast the
recombinant protein's strycture, but found that confidence drops in the flexible linker region instead
of functional domains, which indicated that their structures had been altered. Nevertheless, the
relevant articles wrote that the fusion protein could serve expected function (Figure 31). Hence, it was
not appropriate to leverage Alphafold to forecast the fusion protein's structure.

![img](https://static.igem.wiki/teams/5855/engineering/figure-31.webp)

Figure 31. Diagonal matrix comparison of BslA-dCBM showing the confidence pridicted by
Alphafold.
a. BslA
predicted result by AlphaFold. b. CBM predicted result by AlphaFold.

With the usage of Kabsch
Comparison, it could be told that CBM domains (cbm*1, cbm*2) showed minimal displacement (RMSD <
1.1 Å) and BslA deviated more (4.264 Å) but remained acceptable thanks to linker flexibility indicating
that the fusion protein structure's rationality since all domains' functions remained stable. Meanwhile,
we leveraged GROMACS simulation which also confirmed stable BslA and CBM conformations as the result of
β-structures maintenance and no unfolding or disorder (Figure 32). Other information of the fusion
protein's structure prediction is available in Model page.

[The Model page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27mathematical-modeling%27%29%20%7D%7D)

![img](https://static.igem.wiki/teams/5855/engineering/figure-32.webp)

Figure 32. GROMACS simulation results of BslA-dCBM for 3 ns.

We examined the translation of BslA-dCBM by
SDS-PAGE. As is shown in Figure 33, no stronger bonds were found at 34.68 kDa, the relative molecular
mass of BslA-dCBM.

![img](https://static.igem.wiki/teams/5855/engineering/20251008-193858.avif)

Figure 33. SDS-PAGE of expression products of BslA-dCBM.
Lane 1: Protein ladder; Lane
2-4: Whole-cell
lysate, supernatant and pellet from uninduced cells, respectively; Lane 5-7: Whole-cell lysate,
supernatant and pellet from 0.2 mmol/L IPTG induced cells, respectively; Lane 8-10: Whole-cell lysate,
supernatant and pellet from 0.5 mmol/L IPTG induced cells, respectively; Lane 11-13: Whole-cell lysate,
supernatant and pellet from 1 mmol/L IPTG induced cells, respectively.

Hence, we managed to distinguish
the target protein from others by deleting the 6 × His tag before it and to optimize the expression
condition guided by corresponding articles. After the verification with SDS-Page, 1 mmol/L IPTG was
considered the most appropriate concentration for our engineered bacterial to express BslA-dCBM (Figure
34).

![img](https://static.igem.wiki/teams/5855/engineering/figure-34.webp)

Figure 34. SDS-PAGE of expression products of BslA-dCBM after deleting the His tag before.

Lane 1:
Protein ladder; Lane 2-4: Whole-cell lysate, supernatant and pellet from uninduced cells, respectively;
Lane 5-7: Whole-cell lysate, supernatant and pellet from 0.2 mmol/L IPTG induced cells, respectively;
Lane 8-10: Whole-cell lysate, supernatant and pellet from 0.5 mmol/L IPTG induced cells, respectively;
Lane 11-13: Whole-cell lysate, supernatant and pellet from 1 mmol/L IPTG induced cells, respectively.

Afterwards, we decided to purify the hydrophobic protein by IMAC with common methods guided by the
specification whose SDS-PAGE, however, demonstrated relatively shallow bonds after the elution of buffer
with high imidazole concentration (Figure 35).

![img](https://static.igem.wiki/teams/5855/engineering/figure-35.webp)

Figure 35. SDS-PAGE of expression products of BslA-dCBM purified by IMAC using traditional
methods.
Lane
1: Protein marker; Lane 2: BslA-dCBM supernate after being bound to Ni-NTA resin; Lane 3: BslA-dCBM
supernate eluted with Buffer A; Lane 4-7: BslA-dCBM supernate eluted with 50, 150, 300 and 500 mmol/L
imidazoles.

#### Learn

Successful as the construction of pET-28a(+)-BslA-dCBM-Kan was, the fusion protein's production
efficiency was far below our expectation. Such phenomenon reminded us of the significance of expression
conditions. During the protein expression experiment mentioned above, we added IPTG when
OD600 reached 0.6, incubated the engineered *E. coli* for 16 h as well as resuspened
with 10mL Buffer A (100 mmol/L Tris, 0.5 mmol/L NaCl and 20 mmol/L imidazole), which was effective for
regular protein but obviously unsuitable for our fusion protein. Then, by studying relevant literature,
we ultimately confirmed our final expression protocal, leading to the success of the acquisition of
BslA-dCBM. Meanwhile, since intracellular proteins was suspected to disturb the normal bonds formed by
the recombinant protein, we learnt that the size of the target protein should be under considerartion
while designing a plasmid, which was able to save a great deal of time in the following experiment. With
the ultimate success in protein expression, we attempted to depurate BslA-dCBM but failed since no
strong bonds were found at high immidazole concentration. Hence, new protocols were carried out.

### Loop 2

#### Design

As the functional testing of BslA-dCBM was scheduled, the pure protein solution was of tremendous
necessity. Hence, we attempted to adopt Immobilised Metal Affinity Chromatography (IMAC) to realize the
purification process with the help of Ni-NTA resin and dialysis to remove immidazole impurity from the
elution solution.

#### Build

The usage of traditional IMAC ways to purify the recombinant protein had proved invalid, from which we
suspected that the His tag of BslA-dCBM failed to integrate with nickel. Therefore, from scientists'
experience and depuration approaches mentioned in assays, we discussed and summarized two strategies to
enhance the binding of our protein and nickel. For one, before the elution of imidazole, protein samples
could be incubated at 4°C for several hours. For another, we let the supernatant interact with the
Ni-NTA
resin 5 times.

#### Test

As depicted in Figure 31, without optimition, there emerged two bands at 50 mmol/L imidazole buffer which
could be explained by following theories. To begin with, we supposed that another protein was also
equipped with structures similar to His tag that enabled it to flow through the resin with imidazole
solution. Plus, as 50 mmol/L was not a proper concentration for the elution of regular proteins, the
poor combination of the protein and nickel in the Ni-NTA resin could also lead to the generation of
multiple bands in SDS-PAGE. Thus, an inspection was conducted to find whether there existed protein
impurity and ended with a negative result. From this, we designed a novel scheme to let BslA-dCBM
integrate well with nickel. After leveraging new protocols to depurate, we conducted an SDS-PAGE
experiment, from which a band near 36 kDa was discovered when BslA-dCBM was eluted with 300 mmol/L
imidazoles (Figure 36).

![img](https://static.igem.wiki/teams/5855/engineering/figure-36.webp)

Figure 36. SDS-PAGE of expression products of BslA-dCBM purified by IMAC.
a. BslA-dCBM
purified after 4
hours of incubation. Lane 1: Protein ladder; Lane 2-3: Supernatant and pellet from 1 mmol/L IPTG induced
cells, respectively; Lane 2: BslA-dCBM supernate after being bound to Ni-NTA resin; Lane 3: BslA-dCBM
supernate eluted with Buffer A (100 mmol/L Tris, 0.5 mmol/L NaCl and 20 mmol/L imidazole); Lane
4-7:BslA-dCBM supernate eluted with 50, 150, 300 and 500 mmol/L imidazoles. b. BslA-dCBM flowing 5 times
through the Ni-NTA resin. Lane 1: Protein ladder; Lane 2-4: Whole-cell lysate, supernatant and pellet
from 1 mmol/L IPTG induced cells, respectively; Lane 5: BslA-dCBM supernate after being bound to Ni-NTA
resin; Lane 6: BslA-dCBM supernate eluted with Buffer A; Lane 7-10: BslA-dCBM supernate eluted with 50,
150, 300 and 500 mmol/L imidazoles.

Nonetheless, BslA-dCBM possessed a relative molecular mass of 30.63
kDa twice. Such errors made it difficult to judge whether the purified protein was the target one.
Therefore, we conducted a Western Blot (Figure 37), demonstrating that we had acquired BslA-dCBM
engineered with 6 × His Tag, thus initiating the following large-scale depuration procedure.

![img](https://static.igem.wiki/teams/5855/engineering/figure-37.webp)

Figure 37. Western Blot of expression products of BslA-dCBM purified by IMAC.
 Lane
1-4: BslA-dCBM
supernate eluted with 500, 300, 150 and 50 mmol/L imidazoles; Lane 5: BslA-dCBM supernate eluted with
Buffer A (100 mmol/L Tris, 0.5 mmol/L NaCl and 20 mmol/L imidazole); Lane 6: BslA-dCBM supernate after
being bound to Ni-NTA resin.

Eventually, dialysis of the pure proteins acquired from two depuration
approaches took place, where both stategies showcased stong bonds in SDS-PAGE testing (Figure 38), after
which we leveraged BCA assay to measure its concentrationand and was found to be 0.025 mg/mL.

![img](https://static.igem.wiki/teams/5855/engineering/figure-38.webp)

Figure 38. SDS-PAGE of expression products of BslA-dCBM after dialysis overnight.
Lane
1: Protein ladder;
Lane 2: BslA-dCBM purified after 4 hours of incubation after dialysis overnight; Lane 3: BslA-dCBM
flowing 5 times through the Ni-NTA resin after dialysis overnight.

#### Learn

The complicated purification process suggested that distinct samples should be disposed differently while
discussion was necessary for a complete protocal. Additionally, repetitive errors may not be the result
of operation mistakes, but could be attributed to objective causes such as the lack of precision of
protein ladders at low relative molecular mass, which would be resolved with another method to assist,
hence verifying the scientificity of the experiment. The obtainment of BslA-dCBM led us to the final
testing of its waterproof capacity.

### Loop 3

#### Design

After the acquisition and purification of our recombinant protein, we discovered a convenient way to
verify its hydrophobicity through browsing a great number of articles with the usage of red ink and
filter paper[10]. Similar to our BC, filter paper is composed of cellulose with a mass of
hydroxyls, a hydrophilic radical binding well with water and red ink. Meanwhile, the capillary action
makes red ink possible to permeate the paper, whereas the repulsive force generated by BslA-dCBM resists
ink diffusion, which means that the rate of areas without or with the protein is able to test its
hydrophobicity.

#### Build

500 μL of dialysis buffer, TyrBm solution and BslA-dCBM solution were pipetted in the centre of the
circular filter paper (110 mm diameter) and 1 mL of red ink was used to flood the filter paper.
Afterwards, we took pictures of the dyed filter paper and calculated its hydrophobicity ratio:
$$\text{Hydrophobicity Ratio}=\frac{\text{Red ink coloured area without the target protein}}{\text{Red
ink coloured area with the target protein}}$$

#### Test

As was planned, the testing procedure came off but ended with a meaningless consequence since the whole
filter paper was stained red. In our following experiments, we increased our yield, diluted the red ink
with deionized water and decreased the volume of dyed water we leveraged, leading to the final success
(Figure 39). Details of our new protocol are elaborated in Results.

![img](https://static.igem.wiki/teams/5855/result/figure-39-1008-2157.webp)

Figure 39. Waterproof ability test of BslA-dCBM with controls.
a. Red ink dripped on
the boundary of
dialysis buffer. b. Red ink dripped on the boundary of 0.079 mg/mL TyrBm. c. Red ink dripped on the
boundary of 0.079 mg/mL BslA-dCBM. d. Histogram of different treatments' hydrophobicity ratio.

Although
the functional testing received success, we were still curious whether such quantity of CBM could
achieve the best effect, thus conducting another round of mathematical modeling. This time, we used
GROMACS molecular dynamics simulation so as to find out the number of CBMs that achieved the strongest
binding of the recombinant protein to BC. The binding force was assessed with three parameters:
hydrophobic area, RMSD and structural analysis. Hydrophobic area analysis demonstrated that BslA with 2
or 3 CBMs possessed relatively more area, meaning they were equipped with better waterproof ability
(Table 7).

Table 7. Proportions of hydrophobic area of BslA-CBMs.

| Quantity of CBM | Number of binding sites | Proportion of hydrophobic area |
| --- | --- | --- |
| 1 | 37 | 55.97% |
| 2 | 47 | 58.18% |
| 3 | 50 | 58.22% |
| 4 | 23 | 58.76% |

In addition, RMSD was also calculated. The lower RMSD is, the steadier the structure of the protein is.
In Figure 40, we could tell that when the quantity of CBM was 2 or 3, the fusion protein possessed good
stability.

![img](https://static.igem.wiki/teams/5855/engineering/figure-40.webp)

Figure 40. RMSD Values of BslA-CBMs during 3 ns molecular dynamics simulation.

Eventually, structural
analysis with PyMOL demonstrated that three CBMs spanned both sides of the cellulose plane, which
collided with our goal of covering only one side of the cellulose but the dCBM matched well with our
goal (Figure 41). If you are interested to learn more, please visit the Model page.

[The Model page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27mathematical-modeling%27%29%20%7D%7D)

![img](https://static.igem.wiki/teams/5855/engineering/20251008-214749.webp)

Figure 41. Structure diagram of two and three CBMs.
a. Two CBMs bind to cellulose. b. Three CBMs bind to cellulose.

#### Learn

The failure of the first trial suggested two drawbacks in our waterproof ability testing protocol.
Firstly, the low production made the fusion protein hard to perform extraordinary hydrophobicity, which
was also proved in articles as the well-performed waterproof coat they experimented on reached 10 mg/mL,
far larger than BslA-dCBM we produced. Secondly, the red ink we used possesses a powerful dyeing
capability, thus exerting stronger force than the repulsion generated by waterproof fusion protein.

## References

* [1]Augimeri R V, Strap J L. The phytohormone ethylene enhances
  cellulose production, regulates CRP/FNR*Kx* transcription and causes differential
  gene expression within the bacterial cellulose synthesis operon of *Komagataeibacter
  (Gluconacetobacter) xylinus* ATCC 53582[J]. Frontiers in Microbiology, 2015, 6: 1459.
* [2]Al-Janabi S S, Shawky H, El-Waseif A A, et al. Stable,
  efficient, and cost-effective system for the biosynthesis of recombinant bacterial cellulose in *Escherichia
  coli* DH5α platform[J]. Journal of Genetic Engineering and Biotechnology, 2022a, 20(1): 90.
* [3]Walker K T, Li I S, Keane J, et al. Self-pigmenting
  textiles grown from cellulose-producing bacteria with engineered tyrosinase expression[J]. Nature
  Biotechnology, 2025, 43(3): 345-354.
* [4]Shuster V, Fishman A. Isolation, cloning and
  characterization of a tyrosinase with improved activity in organic solvents from *Bacillus
  megaterium*[J]. Journal of Molecular Microbiology and Biotechnology, 2009, 17(4): 188-200.
* [5]Jiang S, Yuan J F, et al. Genetically encoded biosensors
  for constrained biological functions in probiotic *Escherichia coli* Nissle[J]. ACS Synthetic
  Biology, 2025, 14: 296-303.
* [6]Wang X, Chen G Q, et al. Reversible thermal regulation for
  bifunctional dynamic control of gene expression in *Escherichia coli*[J]. Nature
  Communications, 2021, 12: 1411.
* [7]Wang S, Xiang S L, et al. Restriction-based
  multiple-fragment assembly strategy to avoid random mutation during long cDNA cloning[J]. Journal of
  Cancer, 2015, 6: 632-635.
* [8]Chen H F, Yuan J F, et al. Design of thermoresponsive
  genetic controls with minimal heat-shock response[J]. ACS Synthetic Biology, 2024, 13: 3032-3040.
* [9]Gilmour K A, Zhang M, et al. Environmentally conscious
  hydrophobic spray coatings on bacterial cellulose for sustainable and reusable textiles[J]. Journal
  of Cleaner Production, 2025, 514: 145775.
* [10]Gilmour K A, Zhang M, et al. Biofilm inspired fabrication
  of functional bacterial cellulose through ex-situ and in-situ approaches[J]. Carbohydrate Polymers,
  2023, 304: 120482.

{% endblock %}


{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}This is Results page{% endblock %}
{% block page\_content %}

![Ribbon](https://static.igem.wiki/teams/5855/generalimg/bupi.svg)

![Scissors](https://static.igem.wiki/teams/5855/generalimg/jiandao.svg)

Results

"Each measurement a milestone, each figure a step forward. Together our results shape the bridge from
imagination to achievement."
 — NAU-CHINA

![Classroom Scene](https://static.igem.wiki/teams/5855/heroimg/results.webp)

Overview

Cellulose Synthesis Module

Objectives

Main Achievements

More Details

Dyeing Module

Eumelanin Synthesis

Indigo Synthesis

Temperature Regulation Module

Objectives

Main Achievements

More Details

Co-culture Module

Objectives

Main Achievements

More Details

Waterproof Module

Objectives

Main Achievements

More Details

References

![](https://static.igem.wiki/teams/5855/generalimg/lineline.svg)

![指示器](https://static.igem.wiki/teams/5855/generalimg/mvsz-half.webp)

## Overview

To pioneer a greener textile alternative, we constructed five functional modules within *E. coli*
for cellulose synthesis, pigment synthesis, temperature regulation, co-culture stability, and
hydrophobic coating. This allows us to achieve a one-step synthesis of colored bacterial cellulose
through temperature regulation. This achievement marks a significant stride towards a paradigm shift in
the sustainable fashion industry.

## Cellulose Synthesis Module

### Objectives

Bacterial cellulose (BC) is a highly crystalline and mechanically robust biopolymer whose superior
tensile strength, water-holding capacity, and biocompatibility make it attractive for biomedical
materials and sustainable textiles.

The *bcs* operon of cellulose-producing *Komagataeibacter sucrofermentans* species comprises
four structural genes, *bcs*A, *bcs*B, *bcs*C and *bcs*D, that act in concert to
assemble and export BC. Among them, *bcs*A encodes a membrane-associated glycosyltransferase that,
once activated by the second messenger cyclic-di-GMP, transfers glucose from UDP-glucose to a growing
β-1,4-glucan chain. Its periplasmic partner BcsB binds c-di-GMP, stabilizes BcsA, and directs the
nascent polymer across the inner membrane; the BcsAB complex is therefore the essential catalytic core.
BcsC forms an outer-membrane channel for polymer extrusion, while BcsD promotes periplasmic
crystallization into ordered microfibrils.

![Figure 1. Genetic organization of <i>bcs</i> operon<sup>[1]</sup>.  When BcsA (green) is activated by c-di-GMP, it incorporates glucose units into a cellulose chain in the cytoplasm using UDP glucose as a substrate. BcsB (blue) guides the glucan chain through the periplasm; BcsD (orange) crystallizes four glucan chains in the periplasm, and finally, BcsC (gray) exports the BC micro-fibrils into the extracellular space.](https://static.igem.wiki/teams/5855/result/figure-1.webp)

Figure 1. Genetic organization of *bcs* operon[1].
When BcsA (green) is activated by c-di-GMP, it incorporates glucose units into a cellulose chain in the
cytoplasm using UDP glucose as a substrate. BcsB (blue) guides the glucan chain through the periplasm;
BcsD (orange) crystallizes four glucan chains in the periplasm, and finally, BcsC (gray) exports the BC
micro-fibrils into the extracellular space.

Our aim is to engineer *E. coli* to enable it to synthesize BC, thereby addressing the high
pollution problem in the traditional textile industry. To achieve this goal, we need to address two key
issues: first, the successful functional expression of long four key genes in *E. coli*; second,
the optimization of induction conditions to balance protein expression and host metabolism, so as to
maximize BC production.

### Main Achievements

* The BcsABCD was co-expressed in *E. coli* DH5α at 25°C via a dual-plasmid system, with
  visible gelatinous BC observed.
* The optimal induction concentration was determined to be 0.05 mmol/L IPTG, which achieved the
  highest glucose utilization efficiency (synthesis rate: 28.7 mg/(L·h); consumption rate: 13.7
  mg/(L·h)) to support efficient BC synthesis.

### More Details

#### Successfully constructed dual-plasmid system for BC synthesis

Given that the full-length *bcs*ABCD gene cluster is excessively long (9,126 bp), we divided it into
two fragments, *bcs*AB and *bcs*CD, for expression. Specifically, *bcs*AB was assembled
into the pACYCDuet-1 vector to construct pACYCDuet-1-bcsAB (Figure 2a), and *bcs*CD was inserted
into the pQE-60 vector to construct pQE-60-bcsCD (Figure 2b). To address the issue that the original T7
promoter in pACYCDuet-1 cannot drive expression in DH5α, we successfully subcloned the *bcs*AB
fragment with the T5 promoter into pACYCDuet-1 via mediation of the pQE-60 vector. All gene fragments
and vector fragments were amplified by PCR (Figure 2c and 2e) and assembled using In-Fusion cloning
technology. Sequencing confirmed the successful construction of the two recombinant plasmids (Figure
2f and 2g). Subsequently, these two plasmids were co-transformed into *E. coli* DH5α and BL21(DE3).

![Figure 2. Plasmid map, PCR verification and sequencing identification of recombinant plasmids                 pACYCDuet-1-bcsAB and pQE-60-bcsCD. ](https://static.igem.wiki/teams/5855/result/figure-2.webp)

Figure 2. Plasmid map, PCR verification and sequencing identification of recombinant
plasmids
pACYCDuet-1-bcsAB and pQE-60-bcsCD.
 a. Plasmid map of pACYCDuet-1-bcsAB. b. Plasmid map of
pQE-60-bcsCD.
c. 1% agarose gel electrophoresis of PCR-amplified *bcs*AB (4,675 bp) and *bcs*CD (4,451 bp).
M: DNA marker; Lane 1: *bcs*AB; Lane 2: *bcs*CD. d. 1% agarose gel electrophoresis of
PCR-amplified pQE-60 vector (3,423 bp). M: DNA marker. e. 1% agarose gel electrophoresis of
PCR-amplified T5 promoter-bcsAB-lambda t0-terminator (4,915 bp) and pACYCDuet-1 vector (2,276 bp). M:
DNA
marker; Lane 1: T5 promoter-bcsAB-lambda t0-terminator; Lane 2: pACYCDuet-1 vector. f. Sequencing
results of the recombinant plasmid pACYCDuet-1-bcsAB. g. Sequencing results of the recombinant plasmid
pQE-60-bcsCD.

#### Selection of DH5α as the host strain for BcsABCD expression

Engineered bacteria were induced for expression in LB medium at 25°C. Gel-like dispersed structures were
exclusively observed in the DH5α culture 6 h following the addition of 0.05 mmol/L IPTG (Figure 3a). In
contrast, no such phenomenon was detected in the BL21(DE3) culture. SDS-PAGE analysis confirmed that
BcsABCD was expressed in both strains (Figure 3b). This indicated that although BL21(DE3) has the
ability to express the proteins, it might fail to assemble active "catalysis-transport"
complexes-possibly
due to excessive and rapid protein expression leading to misfolding of BcsABCD into inclusion bodies, or
toxicity to the cell membrane structure[2]. Therefore, DH5α was selected for subsequent
experiments.

![Figure 3. Expression of BcsABCD.](https://static.igem.wiki/teams/5855/result/figure-3.webp)

Figure 3. Expression of BcsABCD.
a. BC was detected as gel-like dispersed
structures in DH5α
6 h after 0.05mM IPTG induction. b. SDS-PAGE of expression products of BcsABCD. Lane 1: Protein ladder;
Lane 2-3: DH5α protein samples obtained from induction with 0.05 mmol/L IPTG and from the uninduced
group, respectively. Lane 4-5: BL21 protein samples obtained from induction with 0.05 mmol/L IPTG and
from the uninduced group, respectively.

#### Optimization of inducible expression conditions

To optimize BC production, we tested the effect of different IPTG concentrations (0, 0.05, 0.1, 0.5 and
1.0 mmol/L IPTG) on BC synthesis in DH5α. After 6 h, distinct gel-like dispersed structures were first
observed in the medium with a final IPTG concentration of 0.05 mmol/L, followed by those in the media
containing 0, 0.1 and 0.5 mM IPTG. However, SDS-PAGE analysis showed no significant difference in the
expression levels of BcsABCD among the different IPTG concentrations (Figure 4). However, it was
observed that the expression levels of BcsABCD were relatively higher in the groups induced with 0, 0.05
and 0.1 mM IPTG, but the overall difference among all groups was not significant.

![Figure 4. SDS-PAGE of expression products of <i>bcs</i>ABCD under different induction conditions in                 DH5α.](https://static.igem.wiki/teams/5855/result/figure-4.webp)

Figure 4. SDS-PAGE of expression products of *bcs*ABCD under different induction
conditions in
DH5α.
Lane 1: Protein ladder; Lane 2-6: DH5α protein samples derived from induction with 0, 0.05,
0.1,
0.2, 0.5 and 1.0 mmol/L IPTG, respectively.

Given that *bcs*A catalyzes BC synthesis using
UDP-glucose as a direct substrate, DH5α first converts glucose in the medium into UDP-glucose.
Therefore, changes in the glucose concentration in the culture medium should be correlated with BC
production.

We sampled at 0, 6, 18, and 24 h respectively, determined the glucose concentration in the
medium, and plotted a glucose concentration curve based on these data (Figure 5), aiming to indirectly
calculate the BC synthesis rate.

Notably, for the groups with 0 mmol/L and 0.05 mmol/L IPTG, the glucose
concentration first increased and then decreased over time, and the glucose in the medium barely changed
after approximately 18 h. We used the following formulas to determine the synthesis rate and consumption
rate:

$$\text{Glucose synthesis rate (mg/(L}\cdot\text{h))} = \frac{\text{Glucose concentration|}\_\text{0
h}^\text{6 h}
}{\text{Time interval|}\_\text{0 h}^\text{6 h} }$$
$$\text{Glucose consumption rate (mg/(L}\cdot\text{h))} = \frac{\text{Glucose concentration|}\_\text{6
h}^\text{18 h}
}{\text{Time interval|}\_\text{6 h}^\text{18 h} }$$
Under induction with 0.05 mmol/L IPTG,
glucose was consumed efficiently: its concentration increased at a rate of 28.7 mg/(L·h) within 0–6 h,
followed by consumption at a rate of 13.7 mg/(L·h) between 6–18 h (Figure 5). In contrast, the uninduced
group exhibited a lower glucose utilization rate (synthesis rate: 11.6 mg/(L·h); consumption rate: 6.43
mg/(L·h)), while groups induced with high IPTG concentrations (≥0.1 mmol/L) showed almost no glucose
consumption and no BC formation.

![Figure 5. Changes in glucose concentration during cultivation of DH5α strain under induction with                 different IPTG concentrations.](https://static.igem.wiki/teams/5855/engineering/23456-1.webp)

Figure 5. Changes in glucose concentration during cultivation of DH5α strain under
induction with 0, 0.05, 0.1, 0.5 and 1.0 mmol/L IPTG concentrations.
a. Schematic diagram of glucose
concentration changes in 96-well
plates
at different incubation times and IPTG concentrations. b. Time-dependent curve of glucose concentration
in medium during cultivation of DH5α strain induced with different IPTG concentrations.

These results revealed that 0.05 mmol/L IPTG provided an optimal induction intensity, which not only
drove sufficient BcsABCD expression but also avoided excessive burden on the host's central metabolism,
thereby effectively channeling UDP-glucose toward BC synthesis.

Additionally, the leaky expression of the T5 promoter was severe. When the IPTG concentration exceeded
0.1 mmol/L, the stress from heterologous expression might have triggered severe metabolic disorders,
impairing cell growth and basic metabolic functions, and thus inhibiting BC synthesis.

## Dyeing Module

### Eumelanin Synthesis

#### Objectives

To color bacterial cellulose we had produced, we chose eumelanin to dye. Eumelanin is a natural pigment
with excellent optical property and biocompatibility, showing great potential in biomaterials and
biomedical applications[3, 4]. TyrBm, a tyrosinase from *Bacillus megaterium*, can
catalyze the biosynthesis of eumelanin[5]. TyrBm functions both as a monophenol
mono-oxygenase, converting L-tyrosine to L-DOPA, and as a diphenol oxidase, catalyzing L-DOPA to
dopaquinone, after which eumelanin forms spontaneously in the presence of oxygen[6] (Figure
6). Based on this, we constructed a eumelanin synthesis system that expressed TyrBm, aiming to achieve
*in situ* synthesis of eumelanin by *E. coli* in the culture medium.

![Figure 6. Eumelanin biosynthetic pathway.](https://static.igem.wiki/teams/5855/result/figure-6-1.webp)

Figure 6. Eumelanin biosynthetic pathway.

#### Main Achievements

* We successfully expressed and purified TyrBm, and determined its specific activity along with the
  kinetic parameters.
* By heterologously expressing TyrBm in *E. coli*, we achieved visible eumelanin biosynthesis,
  as evidenced by the culture medium turning black.

#### More Details

**Successful expression of TyrBm**

TyrBm coding sequence was synthesized and seamlessly cloned into the pET-28a(+) vector containing the
high-strength T7 promoter to construct the recombinant plasmid pET-28a(+)-TyrBm and transformed it into
*E. coli* DH5α to amplify it (Figure 7).

![Figure 7. Plasmid map of pET-28a(+)-TyrBm and 1% agarose gel electrophoresis of PCR-amplified                 pET-28a(+)-TyrBm components. ](https://static.igem.wiki/teams/5855/result/figure-7.webp)

Figure 7. Plasmid map of pET-28a(+)-TyrBm and 1% agarose gel electrophoresis of
PCR-amplified
pET-28a(+)-TyrBm components.
a. Plasmid map of pET-28a(+)-TyrBm. b. 1% agarose gel electrophoresis
of
PCR-amplified pET-28a(+) vector (5,326 bp). M: DNA marker. c. 1% agarose gel electrophoresis of
PCR-amplified TyrBm (909 bp). M: DNA marker.

Based on the Sanger sequencing results, we confirmed the
accuracy of the plasmid (Figure 8). So we transformed it into *E.coli* (DE3) to express TyrBm
protein.

![Figure 8. Sequencing results of the recombinant plasmid pET-28a(+)-TyrBm.](https://static.igem.wiki/teams/5855/result/figure8.webp)

Figure 8. Sequencing results of the recombinant plasmid pET-28a(+)-TyrBm.

We tried to express the protein
in *E. coli* BL21(DE3) harboring pET-28a(+)-TyrBm at 37°C for 12 h in LB medium and TB medium, and
analyzed the protein expression result by SDS-PAGE (Figure 9b). We found a protein of the expected size
in induced cells with IPTG but not in uninduced cells. This result indicated that the expression of
TyrBm was successful and could be controlled.

![img](https://static.igem.wiki/teams/5855/result/figure9.webp)

Figure 9. Expression of recombinant TyrBm under TB/LB culture conditions.
a. Color
changes of each group
of cultures. -: No addition of IPTG; +: Addition of IPTG. b. SDS-PAGE of expression products of TyrBm.
Lane 1: Protein ladder; Lane 2-4: Whole-cell lysate, supernatant and pellet from uninduced cells in TB
medium, respectively; Lane 5-7: Whole-cell lysate, supernatant and pellet from uninduced cells in LB
medium, respectively; Lane 8-10: Whole-cell lysate, supernatant and pellet from IPTG induced cells in TB
medium, respectively; Lane 11-13: Whole-cell lysate, supernatant and pellet from IPTG induced cells in
LB medium, respectively.

However, no eumelanin biosynthesis was observed in the LB medium (Figure 9a).
This result is not conducive to the establishment of our ultimate weaving and dyeing system. The
ultimate objective of this eumelanin synthesis system was to achieve eumelanin production in LB medium,
enabling its subsequent integration with the cellulose synthesis system. To this end, we next sought to
optimize the medium.

By comparing the compositions of TB and LB media, we found that TB contains higher
levels of amino acids and metal ions. Based on this observation and supported by previous studies, we
hypothesized that substrates and cofactors in the medium might have influenced enzyme activity and
thereby affect eumelanin biosynthesis. So we next optimize the medium by investigating the influence of
substrates and cofactors on protein expression, the enzymatic activity of TyrBm and eumelanin synthesis.
And we determined kinetic parameters of TyrBm under the optimal conditions to characterize its affinity
toward the substrate.

**Optimization of induction conditions for TyrBm expression**

To determine the effects of induction intensity and substrate addition on the soluble expression of the
protein, TyrBm expression has been induced under different LB media for 12 h (Table 1). After that, the
expression results were collected and analyzed by SDS-PAGE (Figure 10). The results showed that the
soluble expression level of TyrBm was relatively high under two conditions: 0.5 mmol/L IPTG with the
addition of L-tyrosine and Cu2+, and 1 mmol/L IPTG without the addition of L-tyrosine and
Cu2+. Considering that the proportion of soluble protein was higher under 0.5 mmol/L IPTG
with the addition of L-tyrosine and Cu2+ condition. So we chose this condition as the optimal
protein expressing condition.

Table 1. Detailed information of experimental combinations.

| Number | IPTG（mmol/L） | Substrates and cofactors addition (0.5 g/L L-tyrosine + 10 μmol/L CuSO4) |
| --- | --- | --- |
| 1 | 0 | - |
| 2 | 0 | + |
| 3 | 0.5 | - |
| 4 | 0.5 | + |
| 5 | 1 | - |
| 6 | 1 | + |

-: No addition of 0.5 g/L L-tyrosine + 10 μmol/L CuSO4; +: Addition of 0.5 g/L L-tyrosine +
10
μmol/L CuSO4.

![img](https://static.igem.wiki/teams/5855/result/figure-10-2.webp)

Figure 10. SDS-PAGE of expression products of TyrBm.
Lane 1: Protein ladder; Lanes 2–3: Supernatant and
pellet from uninduced cells in LB medium without substrates and cofactors, respectively; Lanes 4–5:
Supernatant and pellet from uninduced cells in LB medium with substrates and cofactors, respectively;
Lanes 6–7: Supernatant and pellet from 0.5 mmol/L IPTG induced cells in LB medium without substrates and
cofactors, respectively; Lanes 8–9: Supernatant and pellet from 0.5 mmol/L IPTG induced cells in LB
medium with substrates and cofactors, respectively; Lanes 10–11: Supernatant and pellet from 1 mmol/L
IPTG induced cells in LB medium without substrates and cofactors, respectively; Lanes 12–13: Supernatant
and pellet from 1 mmol/L IPTG induced cells in LB medium with substrates and cofactors, respectively.

**The effect of substrate and cofactors addition to the culture medium on enzyme activity.**

In the previous section, we determined that the optimal induction strength was 0.5 mM IPTG. To
investigate the effect of substrate and cofactors on enzyme activity under this induction intensity,
expression in the engineered bacteria was induced with 0.5 mmol/L IPTG at 37°C for 12 h under two
culture conditions: LB medium and LB medium supplemented with substrates and cofactors (0.5 g/L
L-tyrosine and 10 µmol/L CuSO4 respectively). Then, Ni-NTA 6FF (His-Tag) prepacked Gravity
column (1 mL) was used for Immobilized Metal Affinity Chromatography (IMAC) to purify the target protein
from the supernatant. Then, we used SDS-PAGE to analyze the results of the purification process. From
Figure 11, observable single sharps in lane 5 and 9 showed that purified target proteins were present in
elution buffer containing 500 mmol/L imidazole.

![Figure 11. SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column.](https://static.igem.wiki/teams/5855/result/figure-11.webp)

Figure 11. SDS-PAGE analysis of protein fractions eluted from the Ni-NTA column.
Lane
1: Protein ladder;
Lane 2-5: TyrBm supernate obtained from LB medium supplemented with L-tyrosine and CuSO4
after being bound to Ni-NTA resin, eluted with 20, 300 and 500 mmol/L imidazole, respectively ; Lane
6-9: TyrBm supernate obtained from LB medium after being bound to Ni-NTA resin, TyrBm supernate eluted
with 20, 300 and 500 mmol/L imidazole, respectively.

We dialyzed the extracted TyrBm for 24 h for
enzymatic activity and kinetic assays.

In a 96-well cell culture plate, we prepared different
concentrations of L-tyrosine or L-DOPA solutions for activity assays. When L-tyrosine was used as the
substrate, the monophenolase activity and kinetic parameters of TyrBm were determined. When L-DOPA was
used as the substrate, the diphenolase activity and kinetic parameters were measured. Dopachrome
formation (ε475=3,600/(mol·cm)) was monitored by measuring the OD475 with a
microplate reader. The OD475 measured within 10 s after enzyme addition was recorded as the
control (A0), and the OD475 measured after 5 min of incubation at 37°C was
recorded as A1. ΔA was defined as A1 − A0, and the amount of dopachrome
produced was subsequently calculated from ΔA (Figure 12). One unit (U) of tyrosinase activity was
defined as the amount of enzyme catalyzing the formation of 1 μmol of dopachrome per minute at 37°C. The
concentration of TyrBm under the two induction conditions (LB medium and LB medium supplemented with 0.5
g/L L-tyrosine and 10 μmol/L CuSO4) was determined using a BCA protein assay kit, and the
specific activities (U/mg) were subsequently calculated based on these values.

Under the LB medium
condition, the specific activities of monophenol monooxygenase and diphenolase were 1,461 U/mg and 4,032
U/mg, respectively, whereas in the supplemented LB medium, the corresponding specific activities were
35,648 U/mg and 199,346 U/mg, respectively (Figure 12). This result showed that the addition of
substrates and cofactors enhanced the monophenol monooxygenase activity by approximately 10-fold and the
diphenolase activity by approximately 100-fold. This result showed that the addition of substrate and
cofactors to the culture medium increased the activity of the purified enzyme significantly.

![Figure 12. The 96 Well Cell Culture Plates of tyrosinase TyrBm used for activity＆kinetic assay and ΔA of                 each well. ](https://static.igem.wiki/teams/5855/result/figure-12.webp)

Figure 12. The 96 Well Cell Culture Plates of tyrosinase TyrBm used for activity＆kinetic
assay and ΔA of
each well.
a. TyrBm purified from LB medium supplemented with L-tyrosine and CuSO4. b.
TyrBm
purified from LB medium. Reaction system incubated at 37°C for 30 min, absorbance measured at 475 nm
(ε475=3,600/(mol·cm))

With the initial rate of dopachrome formation, the enzymatic reaction
rate was obtained, from which the kinetic parameters were calculated.

For kinetic parameter
determination, the enzyme exhibiting the highest specific activity was selected. The data were processed
to generate a Michaelis-Menten curve and a Lineweaver-Burk plot. The calculated Michaelis constant
(Km) and maximum velocity (vmax) of the activity of monophenol monooxygenase were
34.37 μmol/L and 2.487 μmol/(L·min). The calculated Michaelis constant (Km) and maximum
velocity (vmax) of the activity of diphenolase were 540.84 μmol/L and 2.274 μmol/(L·min)
(Figure 13). The Km value of activity of monophenol monooxygenase is lower than activity of
diphenolase, and the vmax value is higher. So that the enzyme exhibited a higher affinity for
L-tyrosine. This indicated that the monophenolase active site of the enzyme has the highest affinity for
tyrosine. And this result contributed the kinetic parameters of this enzyme to the iGEM community.

![Figure 13. The kinetic assay results of TyrBm.](https://static.igem.wiki/teams/5855/result/figure-13.webp)

Figure 13. The kinetic assay results of TyrBm.
a-b. Michaelis-Menten plot of enzymatic
reaction from
tyrosine to dopachrome experiments and from L-DOPA to dopachrome. c-d. Lineweaver-Burk double reciprocal
plot of enzymatic reaction from L-tyrosine to dopachrome experiments and from L-DOPA to dopachrome.

**Optimization of eumelanin synthesis**

TyrBm expressed under different induction conditions (Table 1) and the color change of culture medium was
detected after nurturing *E. coli* BL21(DE3) for 12 h. It was observed that a distinct black color
appeared only in the LB medium supplemented with substrate and cofactors (Figure 14).

Taken together,
the above results demonstrated that substrate and cofactors are necessary in the eumelanin synthesis
process.

![Figure 14. Color changes of cultures in each group (Table 1) after 12 h of TyrBm expression.](https://static.igem.wiki/teams/5855/result/result-14-1.webp)

Figure 14. Color changes of cultures in each group (Table 1) after 12 h of TyrBm expression.

**Optimization of eumelanin synthesis control**

However, similar color change process occurs even without induction, which was embodied by number 2 in
Figure 14. But such situations have not been reported in relevant literature[5] using TB
medium. So in order to confirm whether the same blackening phenomenon occurred under the classic TB
condition, and to determine whether this phenomenon in LB medium was specific to the target protein or
caused by the LB medium, eight experimental combinations were designed (Table 2) : LB or TB medium with
± IPTG (0 or 0.5 mmol/L) and ± substrates and cofactors (0.5 g/L L-tyrosine + 10 μmol/L
CuSO4). We observed that under the non-induced condition (0 mmol/L IPTG) with substrates and
cofactors addition, the LB culture medium turned significantly black, while no blackening was observed
in TB culture (Figure 15).

In Figure 10, no band was observed in the absence of the inducer, which
showed that the unexpected change was not caused by the leakage of TyrBm. And no color change was
observed in the TB medium without the addition of the inducer, which showed that some substances in TB
medium could suppress eumelamin synthesis in the absence of induction.

Table 2. Different expression
conditions.

| Number | Medium type | IPTG (mmol/L) | Substrates and cofactors addition (0.5 g/L L-tyrosine + 10 μmol/L CuSO4) |
| --- | --- | --- | --- |
| 1 | LB | 0 | - |
| 2 | LB | 0 | + |
| 3 | LB | 0.5 | - |
| 4 | LB | 0.5 | + |
| 5 | TB | 0 | - |
| 6 | TB | 0 | + |
| 7 | TB | 0.5 | - |
| 8 | TB | 0.5 | + |

-: No addition of 0.5 g/L L-tyrosine + 10 μmol/L CuSO4; +: Addition of 0.5 g/L L-tyrosine +
10
μmol/L CuSO4.

![Figure 15. Colors of each group (Table 2) of cultures after 12 h of TyrBm expression.](https://static.igem.wiki/teams/5855/engineering/figure-11.webp)

Figure 15. Colors of each group (Table 2) of cultures after 12 h of TyrBm expression.

To better
control the *in situ* synthesis of eumelanin, i.e., to suppress the eumelamin synthesis in the
absence of
induction (Figure 14), different substances from TB medium were supplemented into medium culture
containing substrates and cofactors to build special conditions (Table 3), and the color change was
detected after nurturing 12 h to determine a useful supplement (Figure 16). This result suggested that
accurate regulation of eumelanin synthesis was achieved when glycerol was present. Therefore, glycerol
was identified as an effective supplement.

Table 3. Detailed information of experimental combinations.

| Number | Medium type | IPTG (mmol/L) | 89 mmol/L sodium phosphate buffer | 0.4% (v/v) glycerol | Substrates and cofactors addition (0.5 g/L L-tyrosine + 10 μmol/L CuSO4) |
| --- | --- | --- | --- | --- | --- |
| 1 | LB | 0.5 | - | - | + |
| 2 | LB | 0 | - | - | + |
| 3 | LB | 0.5 | - | + | + |
| 4 | LB | 0 | - | + | + |
| 5 | LB | 0.5 | + | - | + |
| 6 | LB | 0 | + | - | + |

![Figure 16. Culture supernatant obtained without bacteria after induction for 12 h of each group (Table                 3).](https://static.igem.wiki/teams/5855/result/figure-16.webp)

Figure 16. Culture supernatant obtained without bacteria after induction for 12 h of each
group (Table
3).

**Dyeing Time Simulation via PDE Model**

To quantitatively characterize the dyeing behavior of pigments on cellulose and to determine the overall
dyeing duration, a partial differential equation (PDE) model was established by Model to describe the
diffusion and adsorption processes of the dye. The model adopts a dual-pore structure, assuming that
cellulose contains both macro- and micro-pores, which jointly influence dye transport.

The simulation
results are shown below:

![Figure 17. Computational visualization of the PDE algorithm for the quantitative analysis of staining                 time dynamics.](https://static.igem.wiki/teams/5855/engineering/20251008-214242.webp)

Figure 17. Computational visualization of the PDE algorithm for the quantitative analysis of staining
time dynamics.

Based on the simulation outcomes, the effective dyeing duration was determined to be
approximately 37,825 s (≈10.5 h), corresponding to the point at which the pigment concentration
within the cellulose matrix reached equilibrium (Figure 17). Beyond this time, the diffusion flux
approached zero, and the color intensity exhibited minimal further change. This result provides a
quantitative reference for optimizing dyeing parameters in experimental and industrial applications,
ensuring sufficient coloration while avoiding unnecessary processing time.

If you are interested to
learn more, please visit the Model page.

[The Model page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27mathematical-modeling%27%29%20%7D%7D)

### Indigo Synthesis

#### Objectives

Based on our successful establishment of the engineered *E. coli* system for the *in situ*
synthesis
of eumelanin, we have demonstrated the feasibility and advantages of using bacterial enzymatic catalysis
for pigment production. Building upon this foundational work and the principle of biological synthesis,
we turned our attention to another pigment of immense industrial importance material-indigo.

Originating
from *Streptomyces cattleya*, CYP102A catalyzes the regioselective and stereoselective
incorporation of one oxygen atom from molecular oxygen into target substrate molecules, facilitating the
hydroxylation of indole and its subsequent conversion into indigo (Figure 18). Therefore, the primary
objective of this work was to harness CYP102A by constructing and optimizing a heterologous expression
system in *E. coli*. We aimed to harness CYP102A by constructing a heterologous expression system
in *E. coli* and achieve efficient *in situ* biosynthesis of indigo directly in the culture
medium[7].

![Figure 18. Indigo biosynthetic pathway.](https://static.igem.wiki/teams/5855/result/figure-18-01.webp)

Figure 18. Indigo biosynthetic pathway.

#### Main Achievements

We identified the optimal conditions for CYP102A expression and purification, which include a tailored
culture medium supplemented with ALA and reaction substrates, and specific elution/dialysis buffers,
yielding a highly effective preparation of the enzyme.

#### More Details

**Construction of pET-28a(+)-CYP102A plasmid**

First, we constructed pET-28a(+)-CYP102A plasmid to express CYP102A (Figure 19). Then, we transformed it
into *E. coli* DH5α to amplify it.

![Figure 19. The plasmid map of pET-28a(+)-CYP102A and 1% agarose gel electrophoresis of PCR-amplified pET-28a(+)-CYP102A components. ](https://static.igem.wiki/teams/5855/result/figure-19.webp)

Figure 19. The plasmid map of pET-28a(+)-CYP102A and 1% agarose gel electrophoresis of
PCR-amplified pET-28a(+)-CYP102A components.
a. The plasmid map of pET-28a(+)-CYP102A. b. 1% agarose gel electrophoresis of PCR-amplified pET-28a(+)
plasmid (5,227 bp) and CYP102A (3,204 bp).

According to the results of colony PCR and Sanger sequencing (Figure 20), we concluded that the
pET-28a(+)-CYP102A plasmids were constructed successfully.

![Figure 20. Verification of recombinant plasmid pET-28a(+)-CYP102A. ](https://static.igem.wiki/teams/5855/result/figure-20.webp)

Figure 20. Verification of recombinant plasmid pET-28a(+)-CYP102A.
a. 1% agarose gel
electrophoresis of colony PCR of using T7 and T7 ter primers. b. The result of sequencing the CYP102A of
the recombinant plasmids.

**Expression and purification of CYP102A**

Next, to explore how the form of ALA and the culture medium influence CYP102A expression, we expressed
the enzyme in *E. coli* BL21(DE3) using both LB and TB media. We strategically leveraged both: TB
for high-yield protein production and LB to concurrently demonstrate the one-step indigo dyeing
function. The cultures were induced by adding IPTG, ALA, tryptophan, and indole to final concentrations
of 0.02 mmol/L, 0.5 mmol/L, 20 mmol/L and 0.02 mmol/L, respectively. After incubation at 37°C for 21 h,
it was observed that the culture medium failed to exhibit the expected blue (Figure 21a). Then we
analyzed the protein expression result by SDS-PAGE (Figure 21b). We found a protein of the expected size
(113.88 kDa) in induced cells with 0.02 mmol/L IPTG. Our findings demonstrated that while the
supplementation of ALA, in either its free or hydrochloride form, had no significant impact on the
protein yield of CYP102A, the use of TB medium markedly enhanced its expression levels compared to LB
medium. However, the expressed protein failed to catalyze the conversion of the substrate into indigo,
indicating a lack of anticipated enzymatic activity. This discrepancy between successful expression and
absent functionality suggests that proper folding or cofactor incorporation may not have been achieved
under the employed experimental conditions[8].

![img](https://static.igem.wiki/teams/5855/result/figure-21-2.webp)

Figure 21. Expression of recombinant CYP102A under TB/LB culture conditions.
a.
Appearance of the culture medium following 21 h of protein expression. b. SDS-PAGE of expression
products of CYP102A. Lane 1: Protein ladder; Lane 2-4: Whole-cell lysate, supernatant and pellet from
uninduced cells from IPTG induced in LB medium with ALA, respectively; Lane 5-7: Whole-cell lysate,
supernatant and pellet from IPTG induced cells in LB medium with ALA·HCl, respectively; Lane 8-10:
Whole-cell lysate, supernatant and pellet from IPTG induced cells in TB medium with ALA, respectively;
Lane 11-13: Whole-cell lysate, supernatant and pellet from IPTG induced cells in TB medium with ALA·HCl,
respectively.

Having noted reports that imidazole in wash or elution buffers turns the dialysis solution yellow and
abolishes CYP102A activity, we eluted the protein using a buffer with 80 mM imidazole. Then, we used
SDS-PAGE to analyze the results of the purification process. From Figure 22, observable single sharps in
lanes 3 showed that purified target proteins were present in elution buffer containing 80 mmol/L
histidine. The eluents were dialyzed with 50 mmol/L Tris-HCl buffer (pH 8.0) with 10% glycerol to remove
histidine and salt.

![img](https://static.igem.wiki/teams/5855/result/figure-22.webp)

Figure 22. SDS-PAGE of expression products of CYP102A purified by IMAC.
Lane 1: Protein marker; Lane 2: CYP102A supernate after being bound to Ni-NTA resin; Lane 3: CYP102A
supernate eluted with buffer; Lane 4-7: CYP102A supernate eluted with 80 mmol/L hisditine.

**Measurement of CYP102A protein concentration**

We leveraged BCA assay to measure the concentration of CYP102A. First, a standard curve was generated
using the BCA assay kit (Figure 23). According to the regression equation, the protein concentration was
determined to be 0.097 mg/mL, which is sufficient for enzyme activity measurement.

![img](https://static.igem.wiki/teams/5855/result/figure-23.webp)

Figure 23. The standard curve of BCA assay kit.

Next, to measure the kinetic parameter of indole, our reaction mixture contained 1 μmol/L CYP102A, 50
mmol/L Tris buffer, 200 μmol/l NADPH and 20 mmol/L tryptophan. The oxidation rates of NADPH were
measured by monitoring the decrease in absorbance at 340 nm using a SpectraMax iD5 microplate absorbance
readerat 30°C (ε340=6.220/(mmol·cm)). Critically, no enzymatic activity was detected for
CYP102A, as evidenced by the absence of a measurable decrease in absorbance at 340 nm upon cofactor
addition. Despite meticulous adherence to the reported protocol, we were unable to reproduce the
enzymatic activity of CYP102A.

We hypothesized that the instability of key components, particularly ALA
and NADPH, during storage or handling may have compromised the functional assembly of the enzyme's heme
cofactor or its catalytic cycle, leading to the observed lack of activity. This interpretation was
subsequently corroborated through correspondence with the original authors. Future investigations will
focus on optimizing the stability of these labile reagents to achieve robust enzyme function.

## Temperature Regulation Module

### Objectives

Temperature regulation module attaches tremendous significance to our one-step coloured bacterial
cellulose synthesis strategy as it breaks through the restriction brought about by two distinct culture
environments in the two-step approach. The module is compsed of three main parts: FourU, CI857 and PhlF.
All three parts led to cellulose production at 25°C whereas bio-based pigments dyeing at 37°C.

To clearly
validate the module's temperature sensitivity, we designed a circuit with two different fluorescent
proteins (Figure 24). At 25°C, the formation of CI857 dimers and FourU stem loop block the expression of
PhlF repressor and monomeric red fluorescent protein 1 (mRFP1), therefore emitting green fluorescenece
by superfolder green fluorescent protein (sfGFP). Under 37°C, nonetheless, PhlF inhibits PhlF promoter
to
decrease sfGFP but to increase mRFP1[12, 13]. In a word, the red fluorescence intensity was
proportional to dyeing efficiency whereas the green fluorescence intensity stood for bacterial cellulose
production.

![img](https://static.igem.wiki/teams/5855/result/figure24.webp)

Figure 24. The testing circuit of the temperature regulation circuit that can express
proteins with
different fluorescence at distinct temperatures.

### Main Achievements

* We predicted the most appropriate temperature for FourU stem loop to disassemble, which is
  similar to
  the
  37°C we anticipated.
* We confirmed the effect of the combination of FourU, CI857 and PhlF, verifying the
  feasibility of the one-step strategy.

### More Details

#### Mathematical prediction of the dissociation of FourU

FourU, the RNA thermometer, was significant to the temperature regulation module. Specifically, the SD
sequence of RBS (AGGAGG) that binds to the UUUU sequence of the thermometer command the combination of
the ribosome and RBS, exerting direct impact on the transition of cellulose synthesis module and dyeing
module. Thus, we used mathematical modeling to grasp its unchaining temperature.

To begin with, RNA
Structure developed by Mathews Laboratory was adopted to predict secondary structures of FourU at
distinct temperatures, finding that the base pairing between AGGAGG and UUUU remained stable at 310 K
(36.85°C), partially disrupted at 430 K (156.85°C) and disassemble absolutely at 520 K (246.85°C)
(Figure
25).

![Figure 25. Predicted secondary structures from RNA Structure of FourU at different temperatures.](https://static.igem.wiki/teams/5855/result/figure-25.webp)

Figure 25. Predicted secondary structures from RNA Structure of FourU at different
temperatures.

Successful as the simulation was, the temperature was far away from the common culture temperature,
suggesting it only a qualitative forecast.

Therefore, a quantitative analysis was of necessity to verify
whether 37°C was a suitable temperature for the transition in the weaving and dyeing platform.
Whereupon,
we defined a translation initiation potential σ:
$$\sigma=\exp (-\frac{1}{RT}(E\_{total}))$$
In the
formula, R = 1.987 × 10−3 kcal/(mol⋅K) is the Boltzmann constant, T is the absolute
temperature, and Etotal is the total energy barrier submitting to:
$$E\_{total}=E\_{SD}+E\_{tRNA}+E\_{stem}+E\_{4U{binding}}+E\_{coop}$$
With the assistance of
Python, we triumphantly plotted temperature response curve by mapping normalized translation potential
to temperature (Figure 26). As is depicted in the curve, the predicted value of the unchaining
temperature (38.0°C) is close to the setting value (37.0°C) and the deviation may be attributed to the
simplification of parameters such as the ionic environment, indicating that it is reasonable to leverage
37°C as a turning point. Concrete annotations and deduction of these formulas could be seen in Model
part.

[The Model page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27molecular-modeling%27%29%20%7D%7D)

![Figure 26. Temperature response curve of FourU.](https://static.igem.wiki/teams/5855/result/figure26.webp)

Figure 26. Temperature response curve of FourU.

#### Successful construction of pET-28a(+)-CI857-FourU-PhlF-mRFP1-sfGFP plasmid

We scheduled an In-Fusion cloning so as to construct the target 8,347 bp plasmid, thus initially
synthesising pET-28a(+)-CI857-FourU-PhlF-mRFP1 plasmid and *mRFP1-sfGFP* linear fragment
respectively. At first, we wanted to use PCR to directly amplify the 7,587 bp vector but failed despite
a great many trials. Therefore, it was arranged to leverage a multi-fragment strategy, which rendered
the triumphant construction of the temperature-responsed plasmid. Details are expounded in Engineering.

[The Engineering page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)

We then transformed it to *E. coli* DH5α to realize
amplification (Figure 27).

![Figure 27. Plasmid map, PCR verification of the recombinant plasmid                 pET-28a(+)-CI857-FourU-PhlF-mRFP1-sfGFP. ](https://static.igem.wiki/teams/5855/result/figure27.webp)

Figure 27. Plasmid map, PCR verification of the recombinant plasmid
pET-28a(+)-CI857-FourU-PhlF-mRFP1-sfGFP.
a. The plasmid map of pET-28a(+)-BslA-dCBM. b. 1% agarose
gel
electrophoresis of the PCR amplified sfGFP (799 bp). c. 1% agarose gel electrophoresis of the PCR
amplified pET-28a(+)-CI857-FourU-PhlF-mRFP1 fragment 1 (4,310 bp) and pET-28a(+)-CI857-FourU-PhlF-mRFP1
fragment 2 (3,298 bp).

Sanger sequencing confirmed the correct assembly of the recombinant plasmid
(Figure 28).

![Figure 28. Sanger sequencing confirmed the correct assembly of the CI857-FourU-PhlF-mRFP1-sfGFP plasmid.](https://static.igem.wiki/teams/5855/result/figure28.webp)

Figure 28. Sanger sequencing confirmed the correct assembly of the
CI857-FourU-PhlF-mRFP1-sfGFP plasmid.

#### Successful expression of distinct sfGFP signals at different temperatures

We transformed the correct plasmid into *E. coli* BL21(DE3) so as to express fluorescent protein,
after which colonies were inoculated into 5 mL of LB medium and incubated at 37°C with rotation at 250
rpm for 16 h to establish the seed cultures. Subsequently, 50 μL of the cultures were transferred into 5
mL of of LB medium at the same temperature and rotation as mentioned before until their OD600
reached 0.9. Protein expression was then induced by addition of 1 mmol/L IPTG and the cultures were
incubated 25°C, 30°C and 37°C for 34 h. Bacteria at each temperature were prepared in triplicate, and
fluorescence intensity emitted by sfGFP along with OD600 were measured at 6, 9, 12 and 30 h
postinoculation (Figure 29)[12, 14].

![Figure 29. Relative fluorescence intensity over OD<sub>600</sub> at different temperatures with tiny                 standard error.](https://static.igem.wiki/teams/5855/engineering/20251008-205339.webp)

Figure 29. Relative fluorescence intensity over OD600 at different
temperatures.

The standard error at the first few hours might be attributed to the unstable growth when adding IPTG at
first, in spite of which data tended to go smoother. From the line graph, it could be inferred that with
time passing, the discrepancy of sfGFP signal of different temperatures expanded, illustrating that our
module realized the expected consequence. To be specific, the sfGFP is placed at the same place of the
Bcs operon anticipated to produce bacterial cellulose at 2°C where the relative fluorescence intensity
mounted to the highest, suggesting the strongest expression of the operon at the temperature. Meanwhile,
we looked forward to beginning the dyeing module when the temperature reaches 37°C. As depicted in the
graph, at 37°C, the sfGFP expressed weakly, while the circuit activated at a lower temperature. Hence,
TyrBm and CYP102A were able to dye without disturbance.

Then, we used the same protocol to test the relative fluorescence
intensity of mRFP1 but demonstrated no discrepancy among different temperatures. Since the sfGFP had
verified the viability of the temperature regulation module, it was figured that the place we put the
fluorescence protein blocked its normal expression. To be specific, in our experiment design, behind the
PhlF repressor lied mRFP1 regulated by the same RBS. It was of possibility that the second gene behind
the RBS expressed relatively poorer than the first. Hence, as long as the repressor expressed normally,
the red fluorescence faded.

In conclusion, the experiments mentioned above verified the feasibility of
our temperature sensitive module.

## Co-culture Module

### Objectives

In previous work, the bacteria cellulose synthesis system and the eumelanin synthesis system were
individually characterized in monocultures. However, it was crucial to determine whether the two systems
could be integrated into the final “weaving and dying system” and function simultaneously. In short, it
was necessary to verify that *E. coli* BL21(DE3) involved in pigment production and DH5α
responsible for cellulose synthesis can coexist within the same culture. In the following sections,
these two strains are referred to as BL21(DE3) and DH5α, respectively.

### Main Achievements

Based on both Wet-Lab experiments and model predictions, we determined that the two strains could coexist
in a single system under a mildly competitive interaction for 20 hours while growing simultaneously. We
also identified the initial inoculation ratio and temperature-switching time that resulted in the
highest yield of dyed bacterial cellulose.

These findings validated the feasibility of constructing a
one-step weaving and dyeing system based on a *E. coli* BL21(DE3)-*E. coli* DH5α co-culture
and provided a foundation for further optimization by indicating the optimal inoculation ratio and
switching time.

### More Details

#### Effect of varying initial inoculation ratios on temporal biomass

Monoculture of BL21(DE3) and DH5α were respectively cultured to the logarithmic phase, and 500 μL of
bacterial mixture was introduced into 50 mL of LB medium with the initial mixing ratios varying from 1:9
to 9:1 (DH5α:BL21(DE3), v/v)[9]. And the co-cultures were cultivated at 25°C for 20 h.
Samples were collected from each of the nine cultures at 4-hour intervals over the 20-hour period, and
OD600 was measured separately (Figure 30).

This result showed that in all co-culture
conditions, the OD600 values increased over time and reached their maximum between
approximately 12-16 hours, followed by a slight decline. This indicated that the co-culture system
entered a stationary or decline phase after 12–16 hours, likely due to nutrient depletion or
accumulation of inhibitory metabolites. At a DH5α:BL21(DE3) ratio of 1:9 (red region), the culture
reached the highest OD600 value of about 1.66 around 20 hours. As the proportion of DH5α
increased (from 2:8 to 9:1), the maximum OD600 values gradually decreased (from around 1.66
down to about 1.05). Which suggested that BL21(DE3)-dominant cultures grew faster and achieved the
greatest biomass and BL21(DE3) contributes more significantly to the overall biomass. This result is
likely due to the faster metabolic rate and of *E. coli* BL21(DE3), which enables it to achieve
higher biomass.

![Figure 30. OD<sub>600</sub> values of different initial inoculation ratios (DH5α:BL21(DE3), v/v) at                 various time points.](https://static.igem.wiki/teams/5855/result/figure-45.webp)

Figure 30. OD600 values of different initial inoculation ratios
(DH5α:BL21(DE3), v/v) at
various time points.

#### Effect of initial inoculation ratios on the temporal abundance of two strains

To determine the relative proportions of the two strains at each sampling point, genomic DNA was
extracted from each sample, and then absolute quantitative real-time PCR[10,11] using a sets
of primers and probes (Table 4) specifically targeting unique genomic sequences of BL21(DE3) was used to
calculate the relative proportions.

We added 100 ng purified genomic DNA of each sampling point into
each 20 μL real-time PCR system containing 0.3 μmol/L of each primer and 0.2 μmol/L probe. After
applying the amplify program (initial denaturation at 95 °C for 2 min, followed by 40 cycles of
denaturation at 95 °C for 15 s, annealing at 59 °C for 30 s, and extension at 72 °C for 30 s) on such
0.2 mL 96-well PCR plate, we calculated the copy number of genomic DNA of BL21(DE3) in each hole and
then the relative abundance of BL21(DE3) was determined (Figure 31).

This result showed that despite the
different initial inoculation ratios, BL21(DE3) maintained dominance in the co-culture system after 4 h
of cultivation among most groups. This may be due to the relatively slow growth rate of DH5α which had
been transformed with two large-sized plasmid.

Since the designed production process involved
synthesizing bacterial cellulose by DH5α during the first 16–20 h, and was followed by heating to kill
DH5α and activate the BL21(DE3)-based pigment synthesis, it was preferable to maintain a similar
proportion of the two strains and avoid the dominance of either one during the initial 20 h of
co-cultivation. This balance ensured sufficient cellulose production while providing adequate biomass of
BL21(DE3) for pigment synthesis after the temperature increase. Based on data in Figure 31, for the
group with an initial ratio of 3:7, the abundance of BL21 was too low at 12 h and remained low after
that. For the group with an initial ratio of 1:9, the abundance of BL21 was excessively high during the
first 8 hours and at 20 h. For the group with an initial ratio of 4:6, the overall trend remained
stable, with the abundances of both strains becoming comparable during the period from 16 h to 20 h. So
we chose 4:6 (DH5α:BL21(DE3), v/v) as the optimal initial inoculation ratio.

Table 4. Primers and probes
used in real-time PCR.

| Strain | Primer-F (5'-3') | Primer-R (5'-3') | Probe (5'-3') | 3′-terminal modification of the probe | 5′-terminal modification of the probe |
| --- | --- | --- | --- | --- | --- |
| BL21(DE3) | CACACAGGAAACAGCTATGACCATG | GATCGCACTCCAGCCAGCTTT | CACTGGCCGTCGTGGCCCGCAC | 3'BHQ2 | 5'CY5 |

![Figure 31. Relative abundance of BL21(DE3) of different initial inoculation ratios (DH5α:BL21(DE3), v/v)                 at various time points.](https://static.igem.wiki/teams/5855/result/figure-46.webp)

Figure 31. Relative abundance of BL21(DE3) of different initial inoculation ratios
(DH5α:BL21(DE3), v/v)
at various time points.

#### Establishment of models for describing and predicting co-culture dynamics

To determine the optimal initial inoculation ratio and temperature-switching time, we established models
to describe the dynamics of OD600 variation and the interaction between the two strains, and
to predict the yields of bacterial cellulose and pigment under different co-culture and monoculture
conditions. Furthermore, we implemented the model as a software tool for practical use (details are
shown in https://2025.igem.wiki/nau-china/mathematical-modeling). Based on experimental data, our model
predicted that the interaction between the two strains is characterized by mild competition. The
rationality of the model was proved by compared the OD600 variation dynamics curvy generated
by such model with relative data in Wet-Lab as mentioned before (Figure 30, Figure 32).

![Figure 32. Predicted OD<sub>600</sub> variation dynamics curvy. ](https://static.igem.wiki/teams/5855/result/figure-47.webp)

Figure 32. Predicted OD600 variation dynamics curvy.

Next, we used this model to predict the
optimal initial inoculation ratio and temperature-switching time.

In the Wet-Lab's experiments, we
observed that the pigment production was always saturated relative to cellulose synthesis, owing to the
deep color and high dying efficiency of the pigment we selected. So, in order to reduce the waste of the
pigment and increase the yield of bacterial cellulose, we set the weight ratio of cellulose synthesis to
pigment production at 9:1 (Figure 33c). Ultimately, an initail inoculation ratio of 0.682 (approximately
4:6) and a temperature-shift time of 16.1 h were identified as the optimal conditions (Figure 33), and
0.879 g/L cellulose and 1.660 g/L pigment could be obtained (Figure 33d). The optimal inoculation ratio
was similar to the conclusion in Wet-Lab's experiments.

![Figure 33. Results predicted by the model. ](https://static.igem.wiki/teams/5855/result/figure-48.webp)

Figure 33. Results predicted by the model.
a. Prediction result of optimal initial
inoculation ratio. b.
Prediction result of optimal temperature-switching time. c. The set weight ratio of cellulose synthesis
to pigment production. d. Prediction results of the yields of cellulose and pigment.

## Waterproof Module

### Objectives

To endow the coloured bacterial cellulose we produced with hydrophobic capability, we introduced a
biofilm-surface layer protein A with a double cellulose binding module (BslA-dCBM). This recombinant
protein has been proved to connect with the cellulose effectively, which helped us achieve a rapid and
precise synthesis of waterproof protein coat[15].

### Main Achievements

* We successfully expressed and purified the BslA-dCBM protein in *E. coli* BL21(DE3), with a
  final
  yield of approximately 125 mg/L.
* We tested the recombinant protein's function on filter paper with red
  ink meaured by hydrophobicity ratio, which proved its hydrophobicity.
* We conducted mathematical
  simulations and concluded that BslA with the double CBM behaved the best for our project.

### More Details

#### BslA-dCBM structure prediction

Initially, AlphaFold was used to predict the structure of BslA and CBM respectively (Figure 34). BslA
structure prediction yielded ipTM = pTM = 0.69, indicating high confidence. BslA adopts a β-barrel fold,
supporting hydrophobic interactions consistent with its biological role. CBM structure prediction
yielded ipTM = pTM = 0.7, indicating high confidence. CBM adopts a β-sandwich fold, typical of
carbohydrate-binding modules, enabling cellulose recognition and adhesive function.

![Figure 34. BslA and CBM predicted result by AlphaFold. a. BslA predicted result by AlphaFold. b. CBM                 predicted result by AlphaFold. ](https://static.igem.wiki/teams/5855/result/figure30.webp)

Figure 34. BslA and CBM predicted result by AlphaFold.
a. BslA predicted result by
AlphaFold. b. CBM
predicted result by AlphaFold.

Nevertheless, when we leveraged the same approach to forecast the fusion
protein's structure, we discovered that confidence majorly drops in the flexible linker region but not
the functional domains, suggesting the change of their normal structures, which contradicted with the
experiment (Figure 35). Such circumstances manifested that it was not reasonable to leverage Alphafold
to forecast the fusion protein's structure.

![Figure 35. Diagonal matrix comparison of BslA-dCBM showing the confidence pridicted by Alphafold. ](https://static.igem.wiki/teams/5855/result/figure31.webp)

Figure 35. Diagonal matrix comparison of BslA-dCBM showing the confidence pridicted by
Alphafold.

Hence,
as for the stability of the recombinant protein, we gave Kabsch Comparison a trial and found that CBM
domains (cbm*1, cbm*2) show minimal displacement (RMSD < 1.1 Å), while BslA deviates more (4.264 Å)
but remains acceptable given linker flexibility. In a word, the fusion protein's structure is
reasonable with all domains retaining their functions. GROMACS simulation was conducted as well,
which demonstrated stable BslA and CBM conformations since β-structures maintained and no unfolding
or disorder was observed (Figure 36). Detailed procedures were provided in Model page.

[The Model page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27molecular-modeling%27%29%20%7D%7D)

![Figure 36. GROMACS simulation results of BslA-dCBM for 3 ns.](https://static.igem.wiki/teams/5855/result/figure32.webp)

Figure 36. GROMACS simulation results of BslA-dCBM for 3 ns.

#### Successful expression and optimization of BslA-dCBM

We constructed the pET-28a(+)-BslA-dCBM and transformed it into *E. coli* DH5α (Figure 37).

![Figure 37. The plasmid map and PCR verification of pET-28a(+)-BslA-dCBM parts. a. The plasmid map                         of pET-28a(+)-BslA-dCBM. b. 1% agarose gel electrophoresis of the PCR amplified BslA-dCBM (918                         bp) and pET-28a(+) vector (5,323 bp).](https://static.igem.wiki/teams/5855/result/figure33.webp)

Figure 37. The plasmid map and PCR verification of pET-28a(+)-BslA-dCBM parts.

a. The plasmid map
of pET-28a(+)-BslA-dCBM. b. 1% agarose gel electrophoresis of the PCR amplified BslA-dCBM (918
bp) and pET-28a(+) vector (5,323 bp).

With colony PCR and Sanger sequencing proving its accuracy
(Figure 38), *E. coli* BL21(DE3) was leveraged for protein expression.

![Figure 38. Verification of recombinant plasmid pET-28a(+)-BslA-dCBM. a. 1% agarose gel                         electrophoresis of colony PCR of using T7 and T7 ter primers. b. The result of sequencing the                         BslA-dCBM of the recombinant plasmid. ](https://static.igem.wiki/teams/5855/result/figure34-1008-1338.webp)

Figure 38. Verification of recombinant plasmid pET-28a(+)-BslA-dCBM.
a. 1%
agarose gel
electrophoresis of colony PCR of using T7 and T7 ter primers. b. The result of sequencing the
BslA-dCBM of the recombinant plasmid.

Induced by 0, 0.2, 0.5 and 1 mmol/L of IPTG respectively,
bacterial expressed the fusion protein at 30°C for 16 h using LB medium. Bands were all
discovered at 34.68 kDa in SDS-PAGE, which showed no difference at diverse IPTG concentrations
(Figure 39). We could not identify whether our target protein was expressed successfully,
blocking the following purification as it was inferred that the bands of intracellular protein
and BslA-dCBM coincided.

![Figure 39. SDS-PAGE of expression products of BslA-dCBM. ](https://static.igem.wiki/teams/5855/engineering/figure-33.webp)

Figure 39. SDS-PAGE of expression products of BslA-dCBM.
Lane 1: Protein
ladder; Lane 2-4:
Whole-cell lysate, supernatant and pellet from uninduced cells, respectively; Lane 5-7:
Whole-cell lysate, supernatant and pellet from 0.2 mmol/L IPTG induced cells, respectively; Lane
8-10: Whole-cell lysate, supernatant and pellet from 0.5 mmol/L IPTG induced cells,
respectively; Lane 11-13: Whole-cell lysate, supernatant and pellet from 1 mmol/L IPTG induced
cells, respectively.

Thus, we deleted the 6 × His tag before BslA-dCBM sequence in the
pET-28a(+)-BslA-dCBM mentioned above so as to distinguish the target protein by reducing its
relative molecular mass (Figure 40).

![Figure 40. The plasmid map of pET-28a(+)-BslA-dCBM after deleting the His tag before and 1%                         agarose gel electrophoresis of the PCR amplified new pET-28a(+)-BslA-dCBM parts. a. The plasmid                         map of pET-28a(+)-BslA-dCBM after deleting the His tag before. b. 1% agarose gel electrophoresis                         of the PCR amplified BslA-dCBM (918 bp). c. 1% agarose gel electrophoresis of the PCR amplified                         pET-28a(+) vector (5,206 bp). ](https://static.igem.wiki/teams/5855/result/figure-40.webp)

Figure 40. The plasmid map of pET-28a(+)-BslA-dCBM after deleting the His tag
before and 1%
agarose gel electrophoresis of the PCR amplified new pET-28a(+)-BslA-dCBM parts.
a. The
plasmid
map of pET-28a(+)-BslA-dCBM after deleting the His tag before. b. 1% agarose gel electrophoresis
of the PCR amplified BslA-dCBM (918 bp). c. 1% agarose gel electrophoresis of the PCR amplified
pET-28a(+) vector (5,206 bp).

Next, colony PCR and Sanger sequencing showed the accruracy of our
plasmid construction work, suggesting that we had triumphantly deleted the His tag before the
target fragment (Figure 41).

![Figure 41. Verification of recombinant plasmid pET-28a(+)-BslA-dCBM after deleting the His tag                         before. a. 1% agarose gel electrophoresis of colony PCR of using T7 and T7 ter primers. b. The                         result of sequencing the BslA-dCBM of the recombinant plasmid. ](https://static.igem.wiki/teams/5855/result/figure-41.webp)

Figure 41. Verification of recombinant plasmid pET-28a(+)-BslA-dCBM after deleting
the His tag
before.
a. 1% agarose gel electrophoresis of colony PCR of using T7 and T7 ter primers. b.
The
result of sequencing the BslA-dCBM of the recombinant plasmid.

Meanwhile, after a comprehensive
review of the existing literature, we expanded the culturing duration from 16 h to 20 h to
ensure the expression level of BslA-dCBM. In addition, we ultimately determined to use 1 mmol/L
IPTG through gradient tests as a result of the recombinant 30.63 kDa protein's deeper bands
emerge at the supernatant induced by 1 mmol/L IPTG (Figure 42).

![Figure 42. SDS-PAGE of expression products of BslA-dCBM after deleting the His tag before. ](https://static.igem.wiki/teams/5855/engineering/figure-34.webp)

Figure 42. SDS-PAGE of expression products of BslA-dCBM after deleting the His tag
before.
Lane
1: Protein ladder; Lane 2-4: Whole-cell lysate, supernatant and pellet from uninduced cells,
respectively; Lane 5-7: Whole-cell lysate, supernatant and pellet from 0.2 mmol/L IPTG induced
cells, respectively; Lane 8-10: Whole-cell lysate, supernatant and pellet from 0.5 mmol/L IPTG
induced cells, respectively; Lane 11-13: Whole-cell lysate, supernatant and pellet from 1 mmol/L
IPTG induced cells, respectively.

#### Successful purification of BslA-dCBM

Then, we purified the crude protein supernatant by Immobilised Metal Affinity Chromatography
(IMAC) using Ni-NTA resin. To achieve the best purification effect, the procedure was optimized
through 4 h of incubation of BslA-dCBM in the resin before dripping or letting the fusion
protein flow 5 times through the Ni-NTA resin. Both approaches realized the success of
purification at 300 mM imidazole (Figure 43).

![Figure 43. SDS-PAGE of expression products of BslA-dCBM purified by IMAC. a. BslA-dCBM purified                         after 4 h of incubation. Lane 1: Protein ladder; Lane 2-3: Supernatant and pellet from 1 mmol/L                         IPTG induced cells, respectively; Lane 2: BslA-dCBM supernate after being bound to Ni-NTA resin;                         Lane 3: BslA-dCBM supernate eluted with Buffer A (100 mmol/L Tris, 0.5 mmol/L NaCl and 20 mmol/L                         imidazole); Lane 4-7:BslA-dCBM supernate eluted with 50, 150, 300 and 500 mmol/L imidazoles. b.                         BslA-dCBM flows 5 times through the Ni-NTA resin. Lane 1: Protein ladder; Lane 2-4: Whole-cell                         lysate, supernatant and pellet from 1 mmol/L IPTG induced cells, respectively; Lane 5: BslA-dCBM                         supernate after being bound to Ni-NTA resin; Lane 6: BslA-dCBM supernate eluted with Buffer A;                         Lane 7-10: BslA-dCBM supernate eluted with 50, 150, 300 and 500 mmol/L imidazoles. ](https://static.igem.wiki/teams/5855/engineering/figure-36.webp)

Figure 43. SDS-PAGE of expression products of BslA-dCBM purified by IMAC.
a.
BslA-dCBM purified
after 4 h of incubation. Lane 1: Protein ladder; Lane 2-3: Supernatant and pellet from 1 mmol/L
IPTG induced cells, respectively; Lane 2: BslA-dCBM supernate after being bound to Ni-NTA resin;
Lane 3: BslA-dCBM supernate eluted with Buffer A (100 mmol/L Tris, 0.5 mmol/L NaCl and 20 mmol/L
imidazole); Lane 4-7:BslA-dCBM supernate eluted with 50, 150, 300 and 500 mmol/L imidazoles. b.
BslA-dCBM flows 5 times through the Ni-NTA resin. Lane 1: Protein ladder; Lane 2-4: Whole-cell
lysate, supernatant and pellet from 1 mmol/L IPTG induced cells, respectively; Lane 5: BslA-dCBM
supernate after being bound to Ni-NTA resin; Lane 6: BslA-dCBM supernate eluted with Buffer A;
Lane 7-10: BslA-dCBM supernate eluted with 50, 150, 300 and 500 mmol/L imidazoles.

Nevertheless,
stronger bonds were found at more than 36 kDa different from the size of the target protein,
from which we indicated that there existed errors between the protein marker and its real
relative molecular mass. To confirm whether we had managed to accomplish the depuration process,
a Western Blot experiment was introduced and demonstrated that stronger bonds were formed by
BslA-dCBM (Figure 44).

![Figure 44. Western Blot of expression products of BslA-dCBM purified by IMAC. Lane 1-4: BslA-dCBM                         supernate eluted with 500, 300, 150 and 50 mmol/L imidazoles; Lane 5: BslA-dCBM supernate eluted                         with Buffer A (100 mmol/L Tris, 0.5 mmol/L NaCl and 20 mmol/L imidazole); Lane 6: BslA-dCBM                         supernate after being bound to Ni-NTA resin. ](https://static.igem.wiki/teams/5855/engineering/figure-37.webp)

Figure 44. Western Blot of expression products of BslA-dCBM purified by IMAC.
Lane 1-4: BslA-dCBM
supernate eluted with 500, 300, 150 and 50 mmol/L imidazoles; Lane 5: BslA-dCBM supernate eluted
with Buffer A (100 mmol/L Tris, 0.5 mmol/L NaCl and 20 mmol/L imidazole); Lane 6: BslA-dCBM
supernate after being bound to Ni-NTA resin.

Next, we carried out dialysis with a buffer
consisting of 100 mmol/L Tris and 0.5 mmol/L NaCl to remove micromolecules like imidazoles so as
to acquire pure BslA-dCBM (Figure 45).

![Figure 45. SDS-PAGE of expression products of BslA-dCBM after dialysis overnight. Lane 1: Protein                         ladder; Lane 2: BslA-dCBM after dialysis overnight. ](https://static.igem.wiki/teams/5855/result/figure-45fx.webp)

Figure 45. SDS-PAGE of expression products of BslA-dCBM after dialysis overnight.

Lane 1: Protein
ladder; Lane 2: BslA-dCBM after dialysis overnight.

Finally, the rocombinant protein's
concentration was calculated using a BCA assay with 8 standards using the microplate reader with
a result of 0.125 mg/mL.

#### Functional testing

As for functional testing, the BslA-dCBM sample was firstly diluted to 0.079 mg/mL. Afterwards,
500 μL of the protein solution was pipetted in the upper half of circular filter paper (110 mm
diameter) and then 10 μL of 1% Hero Red Ink was employed to stain the filter paper while the
same volume of TyrBm with the same concentration and dialysis buffer (100 mmol/L Tris and 0.5
mmol/L NaCl) were used as controls[16]. Meanwhile, to assess the fusion protein's
waterproof capability, we invented a formula:
$$
\text{Hydrophobicity Ratio}=\frac{\text{Red ink
coloured area without the target protein}}{\text{Red ink coloured area with the target
protein}}
$$
As is shown below, the highest hydrophobicity ratio of BslA-dCBM waterproof coats
demonstrates the effectiveness of the recombinant protein (Figure 46).

![img](https://static.igem.wiki/teams/5855/result/figure-39-1008-2157.webp)

Figure 46. Waterproof ability test of BslA-dCBM with controls.
a. Red ink
dripped on the boundary
of dialysis buffer. b. Red ink dripped on the boundary of 0.079 mg/mL TyrBm. c. Red ink dripped
on the boundary of 0.079 mg/mL BslA-dCBM. d. Histogram of different treatments' hydrophobicity
ratio.

#### Mathematical simulations on different CBM quantity

We supposed that different CBM quantity might affect the satability and hydrophobicity of the
recombinant protein, thus performing mathematical simulations.

After verifying the stability of
BslA-dCBM, GROMACS molecular dynamics simulation took place to find out the number of CBMs to
achieve the strongest binding of the recombinant protein to bacterial cellulose. Firstly, the
proportion of hydrophobic area and the RMSD, a value negatively correlated with the structure's
stability, showed that fusion proteins with 2 or 3 CBMs behaved better theoretically (Table 5
and Figure 47).

Table 5. Proportions of hydrophobic area of BslA-CBMs.

| Quantity of CBM | Number of binding sites | Proportion of hydrophobic area |
| --- | --- | --- |
| 1 | 37 | 55.97% |
| 2 | 47 | 58.18% |
| 3 | 50 | 58.22% |
| 4 | 23 | 58.76% |

![Figure 47. RMSD Values of BslA-CBMs during 3 ns molecular dynamics simulation.](https://static.igem.wiki/teams/5855/result/figure-47-1.webp)

Figure 47. RMSD Values of BslA-CBMs during 3 ns molecular dynamics simulation.

However,
structural analysis in PyMOL showcased that three CBMs span both sides of the cellulose plane,
which collided with our goal of covering one side of the cellulose, making this design
unfeasible (Figure 48).

![Figure 48. Structure diagram of two and three CBMs.](https://static.igem.wiki/teams/5855/engineering/20251008-214749.webp)

Figure 48. Structure diagram of two and three CBMs.
a. Two CBMs bind to
cellulose. b. Three CBMs bind to cellulose.

In conclusion, the double CBM module achieved
the best binding characteristic and its combination of BslA was rather stable. Added with the
wet lab's data, BslA-dCBM was an ideal choice for endowing the waterproof capability of the
cellulose. If you are interested to learn more knowledge of CBMs, please visit the Model page.

[The Model page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27molecular-modeling%27%29%20%7D%7D)

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