iGEM 2023 — Team ouc-haide

{% extends "layout\_notebook.html" %}
{% 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 %}
{% block page\_content %}

# Development Notebook

---

2022

**May 1st:** With the help of advisors, core members started learning Introduction to Synthetic Biology and Molecular Biology.

**August** 23rd: Finished studying Introduction to Synthetic Biology and Molecular Biology.

**September** 1st: Core members began learning about the outstanding iGEM team Wikis.

**October 1st:**

•Decided to establish an independent new iGEM team.

•Began to recruit the team members for the new team.

**October 11th:** Team student leaders successfully reached out to the Principal Investigator(PI).

**October 20th:** Completed the recruitment of most team members.

**October 23rd:** Completed the study of excellent iGEM team Wikis.

**November 23rd:** Team members conducted the first literature review and simulate the project proposal.
![](https://static.igem.wiki/teams/4596/wiki/images/the-team-meeting.png)

Figure 1. The team-meeting

2023 The First Quarter

February

**February 11th:** Concluded the first literature review and discussed the project with the advisors.

March

**March 10th:** Team conducted the second literature review.

**March 27th:** Completed the second literature review.

**March 31st:** Human Practice(HP) group conducted a project-related questionnaire survey targeting fellow students.

2023 The Second Quarter

April

**April 6th:** Team summarized the project and prepared a presentation for discussion with our PI.

**April 7th:** Team engaged in the initial project discussion with our team's PI, Professor Li Chunyang, and OUC-China's PI, Professor Liu Guanglei.

**April 19th:** Initiated collaboration with the iGEM team SUSTech-OCE.
![](https://static.igem.wiki/teams/4596/wiki/images/pimeeting.png)

Figure 2. The project discussion with PI and external advisor

May

**May 5th:** Team student leaders engaged in project discussions with our PI again, and upon approval, commenced gene synthesis.

**May 30th:** Wet Lab group received the synthesized genes.

**May 31st:** Transformed Ncds plasmids, streaked bacteria onto agar plates.

June

**June 1st:** Wet Lab group inoculated bacteria into test tubes.

**June 2nd:** Transferred overnight-cultured tube bacteria into small bottles; utilized IPTG to induce expression in the evening.

**June 3rd:** Lysed bacteria, centrifuged, and conducted protein gel electrophoresis for validation.

**June 4th:** Some members conducted interviews for prospective 2024 OUC-Haide team members.

**June 13th:** Member Hongyue Ma completed the hardware draft.

**June 15th:** Team received the iGEM kit.

**June 20th:** Opened the iGEM kit and distributed badges.

**June 22nd:** Wet Lab group submitted preliminary safety forms.

**June 23rd:** Team selected the Bioremediation track.

**June 24th:** Submitted the project description.

**June 26th:** Wet Lab group began the purification of Ncds protein.

**June 28th:** Validated the activity of formaldehyde dehydrogenase.

2023 The Third Quarter

July

**July 2nd:** Wet Lab group explored plasmid enzyme digestion.

**July 5th:** Artist Yuechen Liu completed summer team uniform shirt design.

**July 8th:** Partial members attended the Conference of China iGEMer Community(CCiC) opening ceremony.

**July 9th:** Member Junteng Sun presented the project at the CCiC conference.

**July 12th:** CCiC conference concluded.

**July 14th:** Member Hongyue Ma discovered certain issues in the hardware; decided to optimize it.

**July 20th:** Completed the design of the second-generation optimized hardware.

**July 23rd:** Optimized the hardware again.

**July 24th:** The laboratory went on vacation.

**July 26th:** Designed the third-generation hardware, but it still had some issues.
![](https://static.igem.wiki/teams/4596/wiki/images/attending-ccic-conference.png)

Figure 3. 2023 OUC-Haide iGEM team in CCiC conference

August

**August 11th:** HP group conducted fieldwork for social research, collected questionnaires on-site.

**August 13th:** Artist Yuechen Liu complete the team flag design.

**August 14th:** The laboratory's vacation ended.

**August 16th:** Team visited a biotech company for hardware design learning and exploration.

**August 17th:** Discussed the necessity of formaldehyde removal with the Director of the Respiratory Department Yuan Dongming.

**August 21st:** Collaborated with the iGEM team OUC-China and organized a lecture on synthetic biology development history.

**August 26th:** Member Hongyue Ma finalized the hardware design.

**August 27th:** HP group completed the design of project-related educational Monopoly cards (SynBioGenius).
![](https://static.igem.wiki/teams/4596/wiki/images/visiting-the-dongxiao-bio-tech-company.png)

Figure 4. Visiting the Dongxiao Bio-Tech Company

![](https://static.igem.wiki/teams/4596/wiki/images/the-game-cards.png)

Figure 5. The SyoBioGenius game cards

September

**September 4th:** Team collaborated with the iGEM team SUSTech-OCE on educational comics.

**September 10th:** Wet Lab group finished the design of the kill switch.

**September 11th:** Member Hongyue Ma began contacting factories to initiate hardware production.

**September 13th:** Visualization group submitted the team's promotion video.

**September 15th:** Wet Lab group commenced the purification of formate dehydrogenase.

**September 16th:**

•Performed NBT testing.

•Member Jing He conducted the meeting with the 2024 OUC-Haide iGEM team members.

**September 17th:** Wet Lab group verified the activity of formic acid dehydrogenase.

**September 18th:** HP group participated in the science communication podcast event hosted by iGEM team ZJUT-iGEM

**September 20th:**

•Wet Lab group submitted the final safety forms.

•Wiki manager Yihong Su began to construct project Wiki.

**September 22nd:**

•Wet Lab group determined the impact of formaldehyde on *E. coli* activity.

•The indutry started the production of hardware.

**September 23rd:** Wet Lab group measured the growth curves of *E. coli*.

**September 27th:** Took team photos.
![](https://static.igem.wiki/teams/4596/wiki/images/hejingmeeting.jpg)

Figure 6. Meeting with 2024 OUC-Haide team members

![](https://static.igem.wiki/teams/4596/wiki/images/teamphoto.png)

Figure 7. The photo of visualization group

![](https://static.igem.wiki/teams/4596/wiki/images/takingphoto.png)

Figure 8. The day of taking team photos

October

**October 4th:** Some members assembled the hardware.

**October 12th:**

•Team submits the project Wiki and other materials needed.

•Begins to prepare Presentation Video.
![](https://static.igem.wiki/teams/4596/wiki/images/the-day-of-hardware-assembly.png)

Figure 9. Hardware assembly

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

* [Overview](#Overview)
* [Project Design](#ProjectDesign)

+ [First Cycle for Project Design](#FirstCycleforProjectDesign)
+ [Second Cycle for Project Design](#SecondCycleforProjectDesign)

* [Experiments](#Experiments)

+ [First Cycle for Experiments](#FirstCycleforExperiments)
+ [Second Cycle for Experiments](#SecondCycleforExperiments)

* [Hardware](#Hardware)

+ [First Cycle for Hardware](#FirstCycleforHardware)
+ [Second Cycle for Hardware](#SecondCycleforHardware)
+ [Third Cycle for Hardware](#ThirdCycleforHardware)
+ [Fourth Cycle for Hardware](#FourthCycleforHardware)

* [References](#References)

![](https://static.igem.wiki/teams/4596/wiki/images/hp-toc-backimg.png)

# Overview

---

The purpose of our project is to remove formaldehyde indoors efficiently and safely. In order to achieve this goal, we should focus on the following three aspects:

* Design: design an efficient formaldehyde removal pathway; address the challenge of maintaining engineered bacteria's normal growth under formaldehyde metabolism stress.
* Experiments: choose appropriate experiments to validate the project design.
* Hardware: design practical device for the utilization of engineered bacteria.

On this Engineering page, we present the design-build-test-learn cycles we went through during the development of the entire project.

# Project Design

---

## First Cycle for Project Design

---

**1. DESIGN**

Our primary objective is to remove the formaldehyde from the air, a task that necessitates our engineered bacteria meeting two essential criteria. Firstly, the bacteria must possess the capacity to express a substantial quantity of enzymes required for the degradation of formaldehyde. Secondly, the bacteria need to maintain normal growth even in the presence of formaldehyde-related stress.

During extensive literature review, we unearthed a formaldehyde metabolic pathway known as the reductive glycine pathway [1]. In this pathway, formaldehyde undergoes a series of transformations: it is initially oxidized into formate, which then undergoes metabolization to 10-formyl-THF. Subsequently, 10-formyl-THF undergoes dehydration, resulting in 5,10-methenyl-THF, followed by reduction to 5,10-methylene-THF. This compound then enters the glycine metabolism pathway via the GCS pathway, ultimately leading to the conversion of glycine. From there, glycine is further processed into serine through de-THF, and serine is subsequently deaminated to pyruvate, entering the TCA cycle for further metabolism (Figure 1).

![](https://static.igem.wiki/teams/4596/wiki/images/eng-1-new.svg)

Figure 1. Formaldehyde metabolism via the reductive glycine pathway

Based on literature research, we found evidence that *E. coli* can thrive in environments with low formaldehyde concentrations. Therefore, we have selected the *E. coli* BL21 strain as a highly suitable candidate for our project.

**2. BUILD**

We opted for the Biobrick RFC [10] standard since the majority of parts available in the Registry database are RFC [10] compatible. This compatibility greatly simplifies the assembly process of our composite parts. We conducted virtual plasmid construction using SnapGene.

**3. TEST**

We have selected the formaldehyde dehydrogenase derived from *Pseudomonas putida*, and its Km value and stability within *Escherichia coli* have been confirmed through an extensive literature review. The research revealed that the Km value for formaldehyde was 67μM, while for NAD+ it was 56μM [2].

**4. LEARN**

After consulting with Professor Guanglei Liu, we realized that the overexpression of enzymes within *Escherichia coli* can adversely affect its viability, especially in environments exposed to formaldehyde stress. As a result, it is advisable to choose a metabolic pathway that requires fewer enzymes for the efficient elimination of formate.

## Second Cycle for Project Design

---

**1. DESIGN**

After another round of brainstorming and a thorough literature review, we decided to retain formaldehyde dehydrogenase for our project. However, for the metabolism of formate, we opted to use formate dehydrogenase to convert formate into carbon dioxide and water. Our inspiration for this change came from several papers published by researchers at the Dalian Institute of Chemical Physics in China.

In their research, they designed and synthesized a novel non-natural cofactor called NCD using biological methods [3], and in addition, they made modifications to formaldehyde dehydrogenase [4], formate dehydrogenase, and ME [5]. Interestingly, formaldehyde dehydrogenase and formate dehydrogenase were found to reduce NCD to NCDH, while ME could oxidize NCDH back to NCD. This innovative approach allowed us to achieve a redox balance in our engineered bacteria, enabling it both survive and efficiently metabolize formaldehyde.

![](https://static.igem.wiki/teams/4596/wiki/images/eng-2-new.svg)

Figure 2. The metabolic pathway of formaldehyde

**2. BUILD**

The establishment of the NCD cofactor involved modifying nicotinic acid mononucleotide adenylyltransferase from *Escherichia coli* through a series of mutations, namely P22K, Y84V, Y118D, C132L, and W176Y. In the case of Ncds-2, the P22K mutation significantly altered the binding pocket for NTP. In the CTP-Ncds-2 complex, the cytosine's amino group formed a hydrogen bond with the oxygen atom of F177, while K22 contributed an additional hydrogen bond with the carbonyl oxygen of cytosine. This interplay of hydrogen bonds, along with steric hindrance in the NTP binding pocket, collectively led to the preference of Ncds-2 for CTP.

Regarding pyridine nucleotide specificity, H45 and W117 in NadD engaged in π-π stacking interactions with the nicotinic ring of N(a)MN, while Y118 formed hydrogen bonds with the carboxyl and amide groups of NaMN and NMN, respectively. Consequently, NadD could accommodate both NaMN and NMN. Conversely, for Ncds-2, no such π-π stacking interactions were observed. The key residue, D118, formed a stronger hydrogen bond with the amide group of NMN, whereas the interaction with the carboxyl group of NaMN was significantly reduced due to electrostatic repulsion. These structural features effectively explained why Ncds-2 exhibited a preference for NMN over NaMN.

We chose the Biobrick RFC [10] standard as it aligned with the majority of parts available in the Registry database, simplifying the assembly process of our composite parts. Virtual plasmid construction was conducted using SnapGene. Meanwhile, the lactose operon enabled us to induce protein expression as needed.

![](https://static.igem.wiki/teams/4596/wiki/images/design-module2.png)

Figure 3. Genetic circuit design of the NCD synthetic biological system

**3. TEST**

We utilized the Autodock4 software to conduct molecular docking studies involving different molecules and proteins, as demonstrated in the [model](https://2023.igem.wiki/ouc-haide/model) interface. The binding energy of the Ncds-2 protein with CTP was determined to be -3.23 kcal/mol, accompanied by a root-mean-square deviation (RMSD) of 2.687 (43 to 43 atoms). In contrast, the binding energy of the Ncds-2 protein with NMN was found to be -5.32 kcal/mol, with an RMSD of 2.327 (30 to 30 atoms).

**4. LEARN**

The metabolic pathway exhibited excellent orthogonality due to the use of the non-natural cofactor NCD, which facilitated the exploration of diversifying this pathway in the past. Ncds-2 and NCD-linked enzymes opened up new opportunities to consider more orthogonal redox chemistry at the metabolic level back then [6]. These developments were conducive to the ongoing efforts involving non-natural bases and non-natural amino acids, which aimed to expand our capacity in terms of understanding and reprogramming life in the past [7].

# Experiments

---

## First Cycle for Experiments

---

**1. DESIGN**

We've selected the pET-28(a) plasmid, which contains a lactose operon, as our gene synthesis vector. Our objective is to study how different IPTG concentrations impact protein expression.

**2. BUILD**

We employed IPTG at final concentrations of 0.2 mM and 1 mM as inducers for protein expression in our study.

**3. TEST**

When inducing with IPTG at concentrations of 0.2 mM and 1 mM, we observed varying expression levels among different proteins. NMN synthetase and Ncds-2 exhibited sufficient expression levels at 0.2 mM IPTG concentration. However, proteins with larger molecular weights, including CTPs, formaldehyde dehydrogenase, formate dehydrogenase, and ME, displayed low expression levels at 0.2 mM IPTG concentration but showed improved expression at 1 mM IPTG concentration.

**4. LEARN**

In the paper titled "Impact of the Expression System on Recombinant Protein Production in *Escherichia coli* BL21," the authors explored the influence of different IPTG concentrations on the expression of the YFP protein within the chosen lactose operon expression system. The findings indicated that the highest level of YFP expression was achieved at an IPTG concentration of 0.1 mM [8]. Notably, YFP is comprised of 239 amino acids and has a molecular weight of approximately 27 kDa, a size similar to that of NMN synthetase and Ncds-2 proteins. Interestingly, this aligns with our discovery that an IPTG concentration of 0.2 mM was optimal for the expression of these two proteins. However, it's important to note that, due to time constraints and a lack of relevant literature, we did not further investigate the hypothesis that larger proteins might necessitate higher inducer concentrations. Subsequent research in our study focused more on formaldehyde metabolism within the cell-free system.

## Second Cycle for Experiments

---

**1. DESIGN**

The in vitro activity validation of Ncds-2, formaldehyde dehydrogenase, formate dehydrogenase, and ME was conducted using HEPES as the buffer system.

**2. BUILD**

In the [experiments](https://2023.igem.wiki/ouc-haide/experiments) we added the relevant enzymes, substrates, and colorimetric reagents in vitro, following the specified procedures.

**3. TEST**

Absorbance measurements were taken at 570nm using a microplate reader to assess enzyme activity. Our observations revealed that the absorbance exhibited significant changes, and these changes occurred more rapidly as the substrate quantity increased (Figure 4).

![](https://static.igem.wiki/teams/4596/wiki/images/newresult.png)

Figure 4. Determination of FalDH and FDH activity

Protein concentration was determined using the Bicinchoninic Acid (BCA) method (Figure 5). Following the fitting process, the concentration of the purified Ncds-2, which was diluted 10 times, was determined to be 0.58 mg/mL.

![](https://static.igem.wiki/teams/4596/wiki/images/eng-f5.svg)

Figure 5. Standard curve for determination of protein content by BCA method

**4. LEARN**

Indeed, Ncds-2 demonstrated its ability to catalyze the synthesis of NCD. Furthermore, formaldehyde dehydrogenase, formate dehydrogenase, and ME were all found to be capable of utilizing NCD.

# Hardware

---

## First Cycle for Hardware

---

**1. DESIGN**

The goal of our project is to eliminate formaldehyde from contaminated air. We intend to modify engineered bacteria to effectively remove formaldehyde; our target application sites include family homes and industrial facilities, necessitating the development of suitable hardware for real-world deployment. For this reason, we have designed a device, featuring an engineering bacteria culture medium at its core. Within this medium, we cultivate the engineered bacteria. Contaminated air will be continuously pumped into this medium, where the engineered bacteria will metabolize the formaldehyde, converting it into harmless carbon dioxide. Ultimately, this process will reduce the formaldehyde concentration to levels below safety standards.

To ensure user-friendliness, we have chosen to employ a solid culture medium. Meanwhile, in order to maximize the contact area between the engineered bacteria and the contaminated air, as well as to promote the growth of the engineered bacteria, we have used a porous cylindrical support structure for the medium. Furthermore, we have ingeniously assembled numerous cylindrical supports together to form the comprehensive support framework for the entire medium. Once securely affixed onto the pillars, this configuration guarantees a substantial air-contact surface area in the end.

![](https://static.igem.wiki/teams/4596/wiki/images/eng-h1.svg)

Figure 6. The first-generation hardware simulation

**2. BUILD**

We utilized a 3D-printed square shell to encase our culture device, as well as employing a 3D-printed support structure for the culture medium. Furthermore, we secured the melted solid medium to the support structure using a specialized mold, inoculating it with engineered bacteria. Afterward, we sealed the device, leaving only the air inlet and exhaust ports open. Contaminated air was then pumped into the device using a small air compressor, creating a controlled environment for our engineered bacteria.

**3. TEST**

The engineered bacteria can grow within the medium, with continuous interaction to contaminated air. However, the growth efficiency of the engineered bacteria in the solid culture medium of this device is quite low, resulting in a minimal population of engineered bacteria. Additionally, contamination of the solid medium by hybrid bacteria has been observed.Furthermore, it has become apparent that the purified air passing out of the culture solution may carry overflowed engineered bacteria from the device, potentially leading to contamination of the surrounding environment.

**4. LEARN**

While using a solid medium offered convenience over a liquid one, it presented a significant challenge in the context of engineered bacteria growth due to its tendency to yield limited growth rates that did not meet the demands for efficient formaldehyde removal. Additionally, addressing the issue of contaminated air entering the system was crucial, as it could lead to unintended contamination by hybrid bacteria. Furthermore, managing the overflow of engineered bacteria in the exhaust air was essential. Unfortunately, our first-generation device was incapable of effectively addressing all these critical aspects.

## Second Cycle for Hardware

---

**1. DESIGN**

After identifying the limitations of our initial hardware, we embarked on a comprehensive hardware improvement effort. Our primary change involved replacing the original solid media with a more efficient liquid media. Simultaneously, we made the decision to incorporate sterilization devices at both ends of the system. This strategic addition ensures that incoming contaminated air, as well as the purified air discharged from the device, remains free from bacterial contamination.

![](https://static.igem.wiki/teams/4596/wiki/images/eng-h2.svg)

Figure 7. The second-generation hardware simulation

**2. BUILD**

We replaced the original 3D-printed incubator, designed for solid media, with a suitable incubator for liquid media and filled it with the liquid medium. On either end of this incubator, we incorporated sealed gas washing bottles containing a 2% NaOH solution. Incoming air is directed through this NaOH solution, effectively sterilizing it by neutralizing any bacteria present as it passes through.

**3. TEST**

The efficiency of the engineered bacteria culture has been significantly improved, with no contamination observed in the culture solution from hybrid bacteria. Moreover, the purified air discharged from the system is free of any surviving engineered bacteria. However, a challenge we've encountered is the issue of an overflow of odor from the engineered bacteria culture solution.

**4. LEARN**

Our second-generation design indeed enhanced the cultural efficiency of engineering bacteria and effectively prevented contamination. Nevertheless, there was a potential concern regarding the odor emitted by the cultured solution of engineered bacteria, which could have been released into the purified air. Hence, it was imperative to address this issue to prevent the introduction of new air pollution.

## Third Cycle for Hardware

---

**1. DESIGN**

After consulting with researchers from Dongxiao Biological Company, we learned that an air filter could effectively remove bacteria from the air. Therefore, we decided to replace the original NaOH gas washing bottle at the inlet with an air filter. This change significantly improved the filtration efficiency, ensuring that the device remained free from contamination. Additionally, we added an odor filter at the outlet of the incubator to eliminate any odors that might escape from the culture solution.

![](https://static.igem.wiki/teams/4596/wiki/images/eng-h3.svg)

Figure 8. The third-generation hardware simulation

**2. BUILD**

We added a small 2.5-inch air filter at the inlet, measuring 17 cm in height and 7.6 cm in width, with a polypropylene microporous membrane internal cartridge. The air compressor was directly connected to the air filter, and the air filter was connected directly to the incubator. At the outlet, we added a 5-inch odor filter, measuring 26 cm in height and 10 cm in width. The odor filter consisted of a housing, a 100-mesh screen, and activated carbon. The outlet of the odor filter leads directly into the NaOH gas washing bottle.

**3. TEST**

The odor filter could effectively adsorb odors, ensuring that the air filtered through the device didn’t carry any unpleasant odors.

**4. LEARN**

Our third-generation device has, for the most part, been successful in ensuring that the engineered bacteria could effectively perform their function of removing formaldehyde. Nevertheless, there were still some shortcomings related to the overall sealing of our device, the arrangement of its various components, and its usability. In order to make it more practical and better suited for real-world applications, we needed to optimize the overall structure of the device.

## Fourth Cycle for Hardware

---

**1. DESIGN**

To make the entire device more practical and suitable for manufacturing, we opted to replace the 3D printed incubator with a fermentation tank. This change allowed us to position the air filter at the inlet and the activated charcoal odor filter at the outlet on top of the fermentation tank, thereby reducing the space occupied. Additionally, we decided to replace the NaOH wash bottle with an air filter due to its short lifespan and the hazards associated with its complex replacement process. By using a relatively higher-pressure air compressor, we ensured that contaminated air could flow smoothly through the unit. Lastly, we incorporated a gooseneck-like exhaust pipe after the odor filter to prevent bacterial contamination at the air outlet. Further details can be found in the [Hardware](https://2023.igem.wiki/ouc-haide/hardware) Section.

![](https://static.igem.wiki/teams/4596/wiki/images/eng-h4.svg)

Figure 9. The fourth-generation hardware simulation

**2. BUILD**

We contacted a factory to manufacture the custom-designed fermenter and an exhaust system. Then, we incorporated essential filtration components into the exhaust system. At both the inlet and outlet, we added compact 2.5-inch air filters measuring 17 cm in height and 7.6 cm in width. Following the air filter at the outlet, we integrated a 5-inch odor filter, sized at 26 cm in height and 10 cm in width. To ensure a consistent flow of contaminated air into the system, we carefully selected a pressure-adjustable air compressor with a 30-liter air tank capacity and a discharge rate of 30 liters per minute.

**3. TEST**

The entire system operates well, ensuring no bacterial contamination and eliminating any odors. Airflow remains constant and stable throughout its operation.

**4. LEARN**

Our fourth-generation device is now well-equipped for practical real-life applications, having taken into account factors such as production costs, operational expenses, efficiency, feasibility, and usage methods. Meanwhile, we are continuously exploring areas for potential optimization in both the device itself and its utilization patterns.

# References

---

[1] 徐蓉, 邓王姝颖, 姜卫红, & 顾阳. (2020). 甲酸生物利用的研究进展. *生物工程学报, 36*(6), 1031-1040.

[2] Xuanliang, L., & Guoren, D. (1997). Qualitative Analysis for a Predator-Prey System with Constant-Rate Prey Harvesting under Type-III Functional Response. *Journal of Biomathematics, 12*(3), 213-222.

[3] Wang, X., Feng, Y., Guo, X., Wang, Q., Ning, S., Li, Q., ... & Zhao, Z. K. (2021). Creating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide. *Nature communications, 12*(1), 2116.

[4] Wang, J., Guo, X., Wan, L., Liu, Y., Xue, H., & Zhao, Z. K. (2022). Engineering Formaldehyde Dehydrogenase from *Pseudomonas putida* to Favor Nicotinamide Cytosine Dinucleotide. *Chembiochem: A European Journal of Chemical Biology, 23*(7), e202100697.

[5] Guo, X., Liu, Y., Wang, Q., Wang, X., Li, Q., Liu, W., & Zhao, Z. K. (2020). Non‐natural Cofactor and Formate‐Driven Reductive Carboxylation of Pyruvate. *Angewandte Chemie International Edition, 59*(8), 3143-3146.

[6] King, E. J., Maxel, S., & Li, H. (2020). Engineering natural and noncanonical nicotinamide cofactor-dependent enzymes: design principles and technology development. *Current Opinion in Biotechnology, 66*, 217–226.

[7] Liu, C. C., Jewett, M. C., Chin, J. W., & Voigt, C. A. (2018). Toward an orthogonal central dogma. *Nature Chemical Biology, 14*(2), 103–106.

[8] Lozano Terol, G., Gallego-Jara, J., Sola Martinez, R. A., Martinez Vivancos, A., Cánovas Díaz, M., & de Diego Puente, T. (2021). Impact of the expression system on recombinant protein production in *Escherichia coli* BL21. *Frontiers in Microbiology, 12*, 682001.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

* [Medium-Preparation](#Medium-Preparation)
* [Protein-Purification](#Protein-Purification )
* [Plasmid-Establishment](#Plasmid-Establishment)
* [BCA-Test](#BCA-Test)
* [Enzyme-Activity](#Enzyme-Activity)

![](https://static.igem.wiki/teams/4596/wiki/images/hp-toc-backimg.png)

# Medium-Preparation

---

# Protein-Purification

---

# Plasmid-Establishment

---

# BCA-Test

---

# Enzyme-Activity

---

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

* [Nicotinamide Cytosine Dinucleotide Cofactor Synthesis](#NicotinamideCytosineDinucleotideCofactorSynthesis)
* [Formaldehyde Metabolic Pathway Construction and Verification](#FormaldehydeMetabolicPathwayConstructionandVerification)
* [Build of Kill Switch](#BuildofKillSwitch)
* [References](#References)

![](https://static.igem.wiki/teams/4596/wiki/images/hp-toc-backimg.png)

# Nicotinamide Cytosine Dinucleotide Cofactor Synthesis

---

We initially employed commercially available competent *Escherichia coli* and introduced the pET-28(a) plasmid, which contains the gene encoding the Ncds-2 protein, into the *E. coli* BL21 strain. Subsequently, all *E. coli* cultures grown in LB medium containing kanamycin were confirmed to contain both the pET-28(a) plasmid and the target gene.

After induction with 0.2 mM IPTG, we collected two sets of *Escherichia coli* cultures: one with the Ncds-2 insert and another without it. Following cell lysis, we measured the intracellular cofactor concentrations. Interestingly, we observed that *E. coli* containing the Ncds-2 gene insert had an NCD concentration of 0.31 mM, while the group lacking Ncds-2 did not exhibit any detectable NCD (Figure 1).

![](https://static.igem.wiki/teams/4596/wiki/images/result-1.svg)

Figure 1.Intracellular cofactor concentrations

(Notably, in the strains capable of synthesizing NCD, the synthesis of NAD was reduced. The NAD concentration was reduced from 0.93 mM to 0.45 mM. This could potentially be attributed to the fact that both NAD and NCD synthesis are dependent on NMN.)

To enhance the efficiency of NCD synthesis while minimizing its impact on intracellular NAD production, we introduced the FtNadE and CtCTPs genes(Figure 2). The enzymes encoded by these genes play a crucial role in catalyzing the precursor molecules NMN and CTP(Figure 3), which are vital for NCD synthesis. Consequently, the inclusion of the FtNadE and CtCTPs genes is considered essential.

![](https://static.igem.wiki/teams/4596/wiki/images/design-module2.png)

Figure 2. The genetic circuit of NCD synthesis

![](https://static.igem.wiki/teams/4596/wiki/images/result-0.png)

Figure 3. The pathway of NCD synthesis

(The synthesis of NCD within the cell has been verified, and we now need to purify the Ncds-2 protein to lay the groundwork for subsequent extracellular experiments.)

We induced our engineered strain to express the target protein and subsequently purified it using a nickel column. Following purification, we performed ultrafiltration and passed the protein solution through a molecular sieve. As a result, we obtained the Ncds-2 protein at a concentration of 5.8 mg/ml. The protein concentration was determined using the Bicinchoninic Acid (BCA) method(Figure 4). Notably, during the measurement process, the purified Ncds-2 protein was diluted tenfold.

![](https://static.igem.wiki/teams/4596/wiki/images/result-3.svg)

Figure 4. Standard curve for determination of protein content by BCA method

# Formaldehyde Metabolic Pathway Construction and Verification

---

To confirm the activity of formaldehyde dehydrogenase, formate dehydrogenase, and malic enzyme, we introduced the respective plasmids into the *E. coli* BL21 strain for induced expression, using the method previously described.

Following the purification of formaldehyde dehydrogenase and formate dehydrogenase, we conducted the procedures(Figure 5) to assess their activity.

![](https://static.igem.wiki/teams/4596/wiki/images/result-4.png)

Figure 5. The protocols of measuring FalDH and FDH activity

The variations in OD values serve as an indicator of dehydrogenase activity. Notably, we observed a more significant change in OD values in the Formate Dehydrogenase (FDH) system compared to the Formaldehyde Dehydrogenase (FalDH) system. As an example, when utilizing NMN and CTP at 5 mM, the change in mOD value per minute in the FDH system was 0.78 higher than that in the FalDH system (Figure 6). This observation suggests that the formate generated during formaldehyde metabolism is rapidly converted into carbonate, effectively reducing its toxicity to the engineered bacteria.

![](https://static.igem.wiki/teams/4596/wiki/images/newresult.png)

Figure 6. The degree of change in OD values increases with the rise in the concentration of NCD synthesis substrates.

In summary, we successfully confirmed the activity of formaldehyde dehydrogenase and formate dehydrogenase in relation to the non-natural cofactor NCD. This achievement has enabled us to construct an orthogonal pathway for the bioconversion of C1 molecules, holding promise for applications in both synthetic and chemical biology research.

To facilitate NCD regeneration, we introduced malic enzyme as a crucial catalyst for the conversion of NCDH to NCD. Furthermore, this enzyme exhibits the capability to synthesize malic acid from carbon dioxide and pyruvate. We have determined the activity of malic enzyme, proceed as Figure 7.

![](https://static.igem.wiki/teams/4596/wiki/images/result-6.jpg)

Figure 7. The protocol of measuring malic enzyme activity

Over time, we observed a gradual increase in the malic acid content(Figure 8). In our system, where NADH is absent, malic enzyme exclusively utilizes NCDH as a cofactor. Hence, the rising malic acid content serves as evidence that malic enzyme is indeed capable of catalyzing the synthesis of malic acid from pyruvate, carbon dioxide, and NCDH.

![](https://static.igem.wiki/teams/4596/wiki/images/result-7.svg)

Figure 8. Reductive carboxylation of pyruvate in vitro

# Build of Kill Switch

---

To ensure that our engineered bacteria does not pose any risk to the environment or users, we implemented a kill switch. We adopted the well-established MazF/MazE toxin/antitoxin system, a robust and widely-used mechanism within iGEM competition.

In our system, the expression of the toxin MazF is governed by the powerful T7 promoter, while the expression of the antitoxin MazE is regulated by the arabinose operon.

![](https://static.igem.wiki/teams/4596/wiki/images/design-module3.png)

Figure 9. The genetic circuit of kill switch

When L-arabinose is introduced into the culture medium, the engineered bacteria commence the expression of the antitoxin MazE. This antitoxin can bind to the constitutively expressed toxin MazF, forming a complex that is non-toxic to cells[1]. Consequently, in a culture medium supplemented with L-arabinose, the engineered bacteria can thrive as usual.

However, if these bacteria inadvertently enter an environment lacking L-arabinose, they will rapidly perish due to the presence of the toxin protein MazF, which prevents the expression of the protective antitoxin MazE. This meticulous design ensures that the engineered bacteria can only survive within a specified environment, thus preventing any potential adverse effects on external ecosystems.

# References

---

[1] Yamaguchi, Y., & Inouye, M. (2011). Regulation of growth and death in *Escherichia coli* by toxin–antitoxin systems. *Nature Reviews Microbiology, 9*(11), 779–790.

{% endblock %}