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).
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.
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.
3. TEST
We utilized the Autodock4 software to conduct molecular docking studies involving different molecules and proteins, as demonstrated in the 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 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).
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.
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.
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.
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.
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 Section.
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
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[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.