iGEM 2023 — Team nau-china

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

## Wet Lab

1. [Mar](#0)
2. [Apr](#0)
3. [May](#0)
4. [Jun](#0)
5. [Jul](#0)
6. [Aug](#0)
7. [Sept](#0)
8. [Oct](#0)

* [Prev](#0)
* [Next](#0)

![](https://static.igem.wiki/teams/4613/wiki/no.png)

1. ## Mar

   Set up NAU-CHINA 2023 team!
2. ## Apr

   brainstormed and read literature in different fields.

   found several directions interested in.

   Consulted relevant experts and selected our project.
3. ## May

   Determined the project--BISTRO.

   Determine E.coli as engineered bacteria.

   Registered our team and determined our team members.
4. ## Jun

   Attended the NIA meeting.

   Carried out meeting with LZU-China.

   Determined cell-free system as our method of implementation.

   Built the gene circuit.
5. ## Jul

   Optimized the design of gene circuit.

   Verified the function of gene circuit.

   Interviewed relevant experts for experiment problems.

   Attended 10th CCiC.
6. ## Aug

   Communicated with NJMU-China face to face.

   Online meeting with LZU-China.
7. ## Sept

   Verified the overall gene circuit and results.

   Completed the improvement part.

   Completed the contribution.

   Completed safety form.
8. ## Oct

   Edited wiki.

## HP

1. [Dec](#0)
2. [Jan](#0)
3. [Apr](#0)
4. [May](#0)
5. [Jun](#0)
6. [Jul](#0)
7. [Aug](#0)
8. [Sep](#0)
9. [Oct](#0)

* [Prev](#0)
* [Next](#0)

![](https://static.igem.wiki/teams/4613/wiki/no.png)

1. ## Dec

   School competition of synthetic biology
2. ## Jan

   Winter training
3. ## Apr

   Mass survey on the awareness of hepatitis and synthetic biology.

   Interview with Prof.Jin Sun

   lnterview with Prof. Xin Yan.·Found partner, established relationships with NJMU-China.

   Designed ICll, invited other teams to participate.
4. ## May

   Interview with Prof.Chen Xi.

   Established relationships with BIT-China.
5. ## Jun

   Interview with Prof.Junbo Chen.

   Survey about the public's intention for home hepatitis test kits.

   Community pet clinic.

   Hosted ICll, had exchanges with our partners and other teams.

   Attended the NIA meeting.

   Established relationships with LZU-CHINA.
6. ## Jul

   Interview with Prof.Ye Ni

   ICII——Popular science training, have exchanges with our partners and other teams.

   Attended 10th CCiC.

   Established relationships with SDU-CHINA.

   Anhui Fuyang city Xingliu town central primary school

   Nanjing Qinhuai District Chaotiangong Street Zhangfuyuan community volunteer teaching
7. ## Aug

   Interview with Prof.Longhai Dai.

   Interview with M.S.Jiayu Wei.

   Education & Communication, had exchanges with our partners and other teams, created wiki of ICll.

   Nanjing Science and Technology Museum.
8. ## Sep

   Interview with Prof.Zengtao Zhong.

   Interview with Dr. YanXin.

   Interview with M.S. Haozhen Zhang and M.S. Junsheng Sheng

   Communication with the startup team.

   Visited the Middle school.

   Visited the Kindergarden.

   Visited the primary school in Nanjing

   Visited the NJAU community.

   Visited the high school.

   Campus activity of iGEM on Yulan Road and carried out the recruitment promotion for college students.
9. ## Oct

   On-campus publicity meeting, summary human practice Content, competition(mini iGEM)

## Model

1. [Apr](#0)
2. [May](#0)
3. [Jun](#0)
4. [Jul](#0)
5. [Aug](#0)
6. [Sept](#0)
7. [Oct](#0)

* [Prev](#0)
* [Next](#0)

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1. ## Apr

   Learned relevant knowledge of synthetic biology, related models and previous projects.
2. ## May

   Determined the direction of modeling.
3. ## Jun

   Selected the models initially and determined the model processing method.
4. ## Jul

   Completed the preliminary establishment.
5. ## Aug

   Adjusted and improved the models.
6. ## Sept

   Completed basic solving of the models.
7. ## Oct

   Completed all modelsand wiki edit.

## Art

1. [May](#0)
2. [July](#0)
3. [Aug](#0)
4. [Spet](#0)
5. [Oct](#0)

* [Prev](#0)
* [Next](#0)

![](https://static.igem.wiki/teams/4613/wiki/no.png)

1. ## May

   Designed the logo of the team.
2. ## July

   Made posters for our project.
3. ## Aug

   Designed the wiki of ICII.

   Directed the Promotional Video.

   Designed ICII online poster.
4. ## Spet

   Composed the wiki and illustrated the principle of the project.

   Designed and made goods and uniforms.
5. ## Oct

   Polished wiki.

## Wiki

1. [Dec](#0)
2. [Jan](#0)
3. [Aug](#0)
4. [Spet](#0)
5. [Oct](#0)

* [Prev](#0)
* [Next](#0)

![](https://static.igem.wiki/teams/4613/wiki/no.png)

1. ## Dec

   School competition of synthetic biology
2. ## Jan

   Winter training
3. ## Aug

   Edited the wiki of ICII

   Constructed the framework of wiki
4. ## Spet

   Edited the wiki.
5. ## Oct

   Edited wiki ＆celebrated wiki freeze!

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ENGINEERING

[* ![](https://static.igem.wiki/teams/4613/wiki/nav-icon/project/description.png)

  Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[* ![](https://static.igem.wiki/teams/4613/wiki/nav-icon/project/engineering.png)

  Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
[* ![](https://static.igem.wiki/teams/4613/wiki/nav-icon/project/protocol.png)

  Protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)
[* ![](https://static.igem.wiki/teams/4613/wiki/nav-icon/project/design.png)

  Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
[* ![](https://static.igem.wiki/teams/4613/wiki/nav-icon/project/implementation.png)

  Implementation](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementation%27%29%20%7D%7D)
[* ![](https://static.igem.wiki/teams/4613/wiki/nav-icon/project/safety.png)

  Safety](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27safety%27%29%20%7D%7D)

* [Loop 1](#Loop1)
  + [Design](#Loop1D)
  + [Build](#Loop1B)
  + [Test](#Loop1T)
  + [Learn](#Loop1L)
* [Loop 2](#Loop2)
  + [Design](#Loop2D)
  + [Build](#Loop2B)
  + [Test](#Loop2T)
  + [Learn](#Loop2L)
* [Loop 3](#Loop3)
  + [Design](#Loop3D)
  + [Build and Test](#Loop3B)
  + [Learn](#Loop3L)
* [Loop 4](#Loop4)
  + [Design and Build](#Loop4D)
  + [Test](#Loop4T)
  + [Learn](#Loop4L)
* [Reference](#Reference)

1

Loop1

### Design

Ochratoxin is another deadly fungal mycotoxin that has attracted widespread attention around the world. In
all kinds of Ochratoxins, Ochratoxin A (OTA) is considered the most toxic, abundant, virulence, toxigenic,
pollutive to agricultural products, and closest relationship with human health. It shows extremely strong
hepatorenal toxicity, teratogenic and carcinogenic effects. The European Food Safety Authority emphasizes
that wine ranks second exposure to OTA. Therefore, we designed this project to detect and degrade OTA.

For OTA detection, currently, methods for detecting OTA consist of instrumental analysis and immunoassay
techniques. Considering the inconvenience of Ochratoxin A (OTA) detection and degradation in the food
industry, we have designed a rapid detection method, detecting by DNA Aptamer. When brewing wine,
manufacturers will try to remove OTA during the fermentation stage. However, there may still be instances
of commercial wines exceeding OTA limits. We found carboxypeptidase A (CPA) derived from bovine pancreas
which could degrade OTA. Considering the practical applicability of the degradation module, we plan to
link CPA with sustainable semi-interpenetrating polymer networks (sIPN). This system was consisted of
elastin-like polypeptides (ELPs), and a pair of reactive protein partners, SpyTag and SpyCatcher, which
could polymerize efficiently under multiple conditions. Then, we encapsulate it in a polymeric
microcapsule consisting of chitosan and sodium alginate. Living sIPN makes the multiple applications of
CPA easier. At the same time, we will use a lysis circuit so that the degradation device could be released
into the liquid.

To check the feasibility of sIPN, we are going to use YFP as reporter gene before.

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/fig-1.jpeg)

Fig. 1 Schematic illustration of sIPN.

### Build

We establish short single-stranded oligonucleotides with high prominent recognition specificity and
affinity to OTA (Fig. 2). G-quadruplexes will form after the binding of aptamer and OTA.

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/fig-2.png)

Fig. 2 The 3D structure of DNA Ampater-OTA complex (from PDB: 7W9N)

To construction the sIPN system, we linked SpyTag or SpyCatcher monomers separately with Hydrophilic
elastin-like polypeptides (ELPs) as backbone (T3 or C3). We obtained the gene through complete synthesis.
Then, we will construct a fusion protein of YFP and T3, so that we can verify this system.

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/fig-3new.jpg)

Fig. 3 Gene circuit of sIPN

### Test

Through literature review, we found that directly using aptamers to detect OTA has low sensitivity, which
might not be able to detect the minimum concentration set by food safety regulations. It does not align
with our application scenario, so we decided to discard this strategy.

We checked the part by SDS-PAGE. As shown in Fig. 4, we expressed and purified T3-YFP and C3 successfully.
Moreover, they are capable of forming the Spy Network.

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/fig-4.jpeg)

Fig. 4 a. The plasmid map of pET29a-T3-YFP. b. The plasmid map of pET29a-C3. c. SDS-PAGE analysis of
the purified protein T3-YFP. d. verification of the fabrication between T3-YFP and C3. If you want to
know more, please click: Rusults

We mixed the bacteria producing T3-YFP and the bacteria producing C3 together and then made immobilized
microcapsules. At the same time, we also attempted to directly use purified T3-YFP to make microcapsule
(Fig. 5 a-2.). After placing two types of immobilized microcapsules at room temperature for 12 hours, we
found that the engineered bacteria and sIPN system will not leak, but small molecules of proteins will
leach out of the microcapsules (Fig. 5 b).

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/fig-5.jpg)

Fig. 5 Image of immobilized microcapsules. a-1. Immobilized microcapsules containing engineered
bacteria with T3-YFP and C3 (0 h); a-2. immobilized microcapsules containing purified T3-YFP protein
(0 h); b-1. immobilized microcapsules containing engineered bacteria with T3-YFP and C3 (12 h); b-2.
immobilized microcapsules containing purified T3-YFP protein (12 h); Scale bar: 1 cm

Moreover, we have established molecular models for T3 and C3, and simulated the protein binding of T3 and
C3. In addition, we have also built a mesh-like molecular model and performed kinetic simulations using
GROMACS, aiming to provide theoretical guidance for subsequent experiments.

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/fig-6.gif)

Fig. 6 The protein structure of C3

In the mathematical model for protein structure determination, we conducted a comparative analysis of
several machine learning methods and ultimately opted for the random forest model to predict the secondary
structure of T3 and C3. The predicted results were found to successfully align with the tertiary
structure, further enhancing the feasibility of protein structure reconstruction.

Table 1. Model Training Metrics of Machine Learning

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/tab-1-fig-6-7.png)

### Test

We require the assistance of addi­tional amplified method for ultrasensitive detection because directly
using aptamers to detect OTA has low sensitivity. We found a locking hairpin recognition-triggered
structure switching strategy, which could drive rolling circle amplification (RCA). The detecting of OTA
could be amplified by this way, and we could achieve more accurate detection.

The protein binding experiment of T3-YFP and C3 shows that the semi-interpenetrating polymer networks
could be applied in practice. We will use this system in the following experiments.

2

Loop2

### Design

For OTA detection, we came up with a strategy that a structure-switching locked hairpin (LHP) triggered
rolling circle amplification (RCA) with circulate template after investigating literatures based on OTA
detection results in the previous loop. If you want more details, you can navigate the site of [the design
page about the sensor module.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)

For degrading enzymes, in order to obtain a CPA with enhanced thermal stability and improved tolerance to
pH variations, we truncated 110 residues from the propeptide and signal peptide to construct mature CPA
(M-CPA). We linked M-CPA into SpyTag monomers, which can be polymerized with SpyCatcher monomers to form a
macromolecular network structure, thereby increasing the stability and sustainable generation of M-CPA.

We need quorum sensing system to control the release of two categories of protein monomers. We used *tra* quorum sensing system provided by Professor Zhong and lysis E protein to construct our autolysis circuit. Bacteria will express lysis E protein abundantly at high density to achieve autolysis.

### Build

We designed and purchased nucleic acid sequences, molecular beacons and magnetic beads needed for OTA
detection.

At the same time, we cloned C3 and T3-M-CPA into the pQE-80L, respectively, constructed pQE-80L-C3 and pQE-80L-T3-M-CPA and we chose *E. coli*  BL21(DE3) as the chassis to express the recombinant protein. And then we constructed a LuxI-LuxR type regulatory system with pSB1C3 and express E protein to periodic release monomers in two categories.

### Test

The result under this detection strategy was still not satisfactory. The trend of florescence is very
chaotic and unstable, which was not in line with our expected results.

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/fig-7.png)

Fig. 7 The response of the circular template method to OTA at varied concentrations (0 pM to 20000
pM). The fluorescence values were detected by fluorescent quantitative PCR instrument.

For T3-M-CPA and C3, we tried various expression conditions to promote their soluble expression, but the soluble expression levels of both were still very low in Fig. 8 and Fig. 9.

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/fi-8.png)

Fig. 8 a. The plasmid map of pQE-80L-C3. b. SDS-PAGE analysis of protein expression trials in *E.
coli* BL21(DE3) cultured in Terrific Broth medium overnight using pQE-80L-C3. The temperature was
20℃. Lane M: protein marker. Lane 1: induced total protein. Lane 2: precipitate. Lane 3: supernatant.
c. SDS-PAGE analysis of protein expression trials in *E. coli* BL21 (DE3) cultured in Terrific
Broth medium for 4 hours using pQE-80L-C3. The temperature was 37℃. Lane M: protein marker. Lane 1:
induced total protein. Lane 2: precipitation. Lane 3: supernatant.

We tested the parts we constructed through sequencing verification. The results showed that we had
successfully constructed the plasmid. Then we tested and described the growth curve of engineering
bacteria. However, the curve indicates a narrow range of oscillation for the system.

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/fig-9.jpg)

Fig. 9 a. The plasmid map of pSB1C3-tra-lysis. b. The results of triple autolysis
groups: pSB1C3-tra-lysis (with IPTG), pSB1C3-tra-lysis (without IPTG), and empty pSB1C3. And we took
samples every 30 minutes.

### Learn

After consulting Associate Professor Junbo Chen, we learned that the circulat template could generate the
higher background due to the occurrence of nonspecific RCA driven by the independent hybridization between
the circulat template and the primer of the LHP. In contrast, the dumbbell template enables both specific
recognition and amplification of the primer sequences, reflected by the better signal to background ratio.
Thus, we changed the circulate template of the strategy into a dumbbell template for OTA detection.

For the expression results of T3-M-CPA and C3, we considered that the low protein expression might be due
to the weak strength of promoter of the pQE vector, so that we cloned C3 and T3-M-CPA into a vector
containing a stronger T7 promoter, respectively.

As shown in Fig. 9 b, our autolysis circuit exhibited poor activity in *E. coli*, and we came up with two hypotheses. The first hypothsis is that the *tra* system in *E. coli* may have a poor ability to lead periodic lysis. And the second hypothesis is that the results can not reflect on the fact due to the poor sample-taking procedures and the low precision of our instruments. If you desire to access our future plan to improve this circuit, click this site: [Project Result Page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27project-result%27%29%20%7D%7D)

3

Loop3

### Design

In our experiments, we observed significant interference from background
fluorescence. After consulting Associate Professor Junbo Chen, we learned that hybridization of
circular templates with LHP primers resulted in non-specific rolling circle amplification (RCA),
inevitably affecting the experimental results. Consequently, based on the information provided by the
professor, we reengineered a dumbbell template for specific primer recognition and
amplification.

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/fig-10.png)

Fig. 10 Schematic illustration of the strategy for OTA detection.

Furthermore, we also discovered that the protein amount expressed by pQE-80L did not meet our
requirements. After a thorough review of the literature, we found that the reducing
environment within *E. coli* BL21 (DE3) adversely affects the formation of disulfide bonds in M-CPA,
leading to poor protein solubility. Consequently, we reconstructed the recombinant vector using pET-29a(+)
and expressed it in SHuffle T7 *E. coli*, which has low reductibility. This modification aimed to
enhance both the efficiency and quality of protein expression.

### Build and Test

Sensor module: In the experiment, we obtained desirable data using the dumbbell template (Fig .11). The fluorescence intensity rises with the increase of concentrations of OTA.

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/fig-11.png)

Fig. 11 The florescence intensity of the qPCR method to detect OTA at varied concentrations (0 pM to
20000 pM).

Degradation module: We constructed two recombinant vectors, pET-29a(+)-T3-M-CPA and pET29a(+)-C3.
Initially, when expressed in *E. coli* BL21 (DE3), we achieved high expression levels.
However, the solubility of T3-M-CPA did not meet the experimental requirements. Considering the impact of the
reducing environment in *E. coli* BL21 (DE3) on disulfide bond formation, we expressed it in SHuffle
T7 *E. coli*, but solubility remained suboptimal.

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/fig-12.jpg)

Fig. 12 a. The plasmid map of pET-29a(+)-T3-M-CPA. b.SDS-PAGE analysis of the purified protein
T3-M-CPA in *E. coli* BL21 (DE3) cultured in LB medium express protein for 12 hours at 20℃. Lane
M: protein marker. Lanes 1-6: flow through and elution containing 10, 20, 50, 100, 100, 250 mM
imidazole, respectively. c. SDS-PAGE analysis of protein expression trials in SHuffle T7 *E.
coli* cultured in 2xYT medium for 12 hours using pET-29a(+)-T3-M-CPA. The temperature was 20℃.
Lane M: protein marker. Lane 1: induced total protein. Lane 2: precipitation. Lane 3: supernatant.

### Learn

Through a review of the literature, we observed that M-CPA has been widespread utility in eukaryotic
expression systems, but its performance in prokaryotic expression systems is less satisfactory. We
hypothesize that this discrepancy may be linked to the formation of inclusion bodies during prokaryotic
expression. The formation of inclusion bodies can be influenced by various factors, such as temperature
and even the correct folding of proteins. These factors may collectively result in solubility issues
encountered with M-CPA. Based on literature, we learned that the solubility issue can be addressed by
adding a SUMO tag before T3-M-CPA.

4

Loop4

### Design and Build

Considering the difficulty of M-CPA expression in prokaryotic, we also found a novel and highly effective OTA degrading enzyme called ADH3, which is from *Stenotrophomonas acidaminiphila*, and then we designed two strategies to solve the problems mentioned above.

In the first strategy, we cloned M-CPA into pET-PC-SUMO vector, and expressed the recombinant protein in SHuffle T7 *E. coli* expression competent cell to reduce the formation of inclusion body. If SUMO-M-CPA can be expressed solubly, we will use TEV hydrolase to cut SUMO off.

In the second strategy, we obtained the plasmid pET46EKLIC\_ADH3 from Associate Professor Longhai Dai of Hubei University. At the same time, we cloned ADH3 into pET-29a(+)-T3-M-CPA vector, constructed pET-29a(+)-T3-ADH3. Both of them were expressed in *E.coli* BL21(DE3).

### Test

In the first strategy, the soluble expression of SUMO-M-CPA (48.5kDa) was slightly increased. Most M-CPA still existed in the form of inclusion bodies.

As for the second strategy, for ADH3, obvious target bands can be seen at 43.4 kDa as shown in Fig. 13 (lanes 4 and 5). For T3-ADH3, obvious target bands can be seen at 73.6 kDa as shown in Fig. 13 (lanes 1 and 2).

![](https://static.igem.wiki/teams/4613/wiki/engineering-success/fig-13.jpg)

Fig. 13 a. SDS-PAGE analysis of protein expression trials in SHuffle T7 *E. coli* cultured in
2xYT medium for 12 hours using pET-29a(+)-SUMO-MCPA. The temperature was 20℃. Lane M: protein marker.
Lane 1: induced total protein. Lane 2: precipitate. Lane 3: supernatant. b. SDS-PAGE analysis of the
purified protein ADH3 in *E. coli* BL21(DE3) cultured in LB medium express protein for 12 hours
at 20℃. Lane M: protein marker. Lanes 1-9: flow through and elution containing 10, 20, 20, 50, 50,
100, 100, 250, 250mM imidazole, respectively. c. SDS-PAGE analysis of protein expression trials in
*E. coli* BL21 (DE3) cultured in LB medium for 12 hours using pET-29a(+)-T3-ADH3. Lane M: protein
marker. Lanes 1-6: flow through and elution containing 50, 50, 20, 20, 10mM imidazole, respectively.

### Learn

According to the result of the above experiment, it is clear that M-CPA is difficult to soluble expression in *E. coli*, although we have taken many strategies to help it. Fortunately, both ADH3 and T3-ADH3 can be expressed solubly, so we chose T3-ADH3 and C3 as the two monomers of the sIPN system.

In conclusion, although the experimental process was very arduous, we finally succeeded in verifying the feasibility of our idea in laboratory environment. At the same time, considering food safety, we will continue to carry out strict regulation of strains to ensure compliance with food safety requirements. In the future, we plan to implement this system in probiotics like *E.coli* Nissle 1917.

5

Reference

Xu G, Zhao J, Yu H, et al. Structural insights into the mechanism of high-affinity binding of
ochratoxin A by a DNA aptamer[J]. Journal of the American Chemical Society, 2022, 144(17):
7731-7740.

Zhang J, Lu Y, Gao W, et al. Structure-switching locked hairpin triggered rolling circle
amplification for ochratoxin A (OTA) detection by ICP-MS[J]. Microchemical Journal, 2023, 186:
108365.

White C E, Winans S C.Identification of amino acid residues of the *Agrobacterium tumefaciens*quorum-sensing regulator TraR that are critical for positive control of transcription[J].Mol Microbiol,2005, 55 (5): 1473-86.

Bernhardt T G, Roof W D, Young R.Genetic evidence that the bacteriophage phi X174 lysis protein inhibits cell wall synthesis[J].Proc Natl Acad Sci U S A,2000, 97 (8): 4297-302.

Xiong L, Peng M, Zhao M, et al. Truncated expression of a carboxypeptidase a from bovine
improves its enzymatic properties and detoxification efficiency of ochratoxin A[J]. Toxins,
2020, 12(11): 680.

Dai L, Niu D, Huang J W, et al. Cryo-EM structure and rational engineering of a
superefficient ochratoxin A-detoxifying amidohydrolase[J]. Journal of Hazardous Materials, 2023:
131836.

Marguet P, Tanouchi Y, Spitz E, et al. Oscillations by minimal bacterial suicide circuits
reveal hidden facets of host-circuit physiology[J]. PloS one, 2010, 5(7): e11909.

Dai Z, Yang X, Wu F, et al. Living fabrication of functional semi-interpenetrating polymeric
materials[J]. Nature Communications, 2021, 12(1): 3422.

![](https://static.igem.wiki/teams/4613/wiki/nav-icon/upbutton.png)

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