Creating New Basic Parts
We synthesized all the genes associated with NCD synthesis and formaldehyde metabolism. Additionally, we conducted in vitro experiments to confirm the activity of these enzymes. And we explored the optimal expression conditions for the enzymes and established purification protocols for the Ncds-2 protein.
- BBa_K4596001
This enzyme synthesizes the non-natural coenzyme nicotinamide cytosine dinucleotide (NCD) using nicotinamide mononucleotide (NMN) and cytidine triphosphate (CTP). It can be used to synthesize NCD, a non-natural coenzyme, for more bioorthogonal reactions. - BBa_K4596002
This enzyme catalyzes the synthesis of cytidine triphosphate (CTP), thereby increasing the CTP content in organisms and providing a crucial precursor for the synthesis of the non-natural coenzyme nicotinamide cytosine dinucleotide (NCD). - BBa_K4596003
This enzyme catalyzes the synthesis of nicotinamide mononucleotide (NMN), raising NMN levels in vivo and providing an essential precursor for the synthesis of the non-natural coenzyme nicotinamide cytosine dinucleotide (NCD). - BBa_K4596004
This enzyme catalyzes the oxidation of formaldehyde to formic acid, and in doing so uses the non-natural coenzyme nicotinamide cytidine dinucleotide (NCD), rather than the nicotinamide adenine dinucleotide (NAD) used by conventional formaldehyde dehydrogenase. - BBa_K4596005
This enzyme catalyzes the oxidation of formic acid to carbon dioxide and water. Notably, it utilizes the non-natural coenzyme nicotinamide cytidine dinucleotide (NCD) instead of the conventional nicotinamide adenine dinucleotide (NAD), showcasing its exceptional orthogonality. - BBa_K4596006
This enzyme catalyzes the synthesis of malic acid from carbon dioxide and pyruvate, and in this process uses the non-natural coenzyme nicotinamide cytidine dinucleotide (NCD), which oxidizes NCDH to NCD, rather than using nicotinamide adenine dinucleotide (NAD).
Hardware Components Available for Reference
After multiple rounds of research, design, construction, testing, and learning (More information can be seen in Engineering Page), we finally designed a dedicated and innovative hardware solution. This device enables our engineered bacteria to effectively remove formaldehyde while preventing any adverse environmental impact (Figure 1).
Our hardware comprises the following components: a fermentation tank, two air filters, an activated carbon odor filter, an air compressor, ventilation pipes, and an anti-pollution exhaust pipe (More information can be seen in Hardware Page). After the polluted gas passes through the whole device, the harmful substance formaldehyde in the polluted air will be effectively removed. Meanwhile, our hardware is unaffected by bacteria in the air and causes no additional contamination.
Air Filter
To maintain sterility when introducing air into the fermentation tank and releasing gas from it, preventing any contamination of the engineered bacterial culture medium, we have implemented a specific solution. We have incorporated small 2.5-inch air filters with dimensions measuring 17 centimeters in height and 7.6 centimeters in width at each inlet and outlet (refer to Figure 3). These filters utilize polypropylene microporous membrane filter elements with a filtration accuracy range from 0.1 microns to 0.5 microns, effectively preventing contamination of the engineered bacterial culture medium.
These air filters have a maximum working temperature of 120°C (at 0.28 MPa), a maximum positive pressure difference of 0.42 MPa (at 25°C), and a maximum back pressure difference of 0.28 MPa (at 25°C). They are characterized by their low-pressure difference, high flow flux, long service life, stable performance, and wide chemical compatibility.
Teams in the future requiring sterile air or aiming to prevent the leakage of engineered bacteria through the exhaust port can consider using the air filters we have employed.
Odor Filter
After passing through the fermentation tank, harmful formaldehyde in the air is removed. However, the odor from the culture medium may enter the room as the gas is discharged. To address this issue, we added a 5-inch odor filter, measuring 26 centimeters in height and 10 centimeters in width, after the air filter connected to the exhaust port. This odor filter comprises a housing, a 100-mesh screen, and activated carbon (Figure 3). We loaded a sufficient amount of activated carbon into the mesh and placed it inverted onto the air filter at the outlet. This process allows the previously sterilized air, after passing through an air filter, to be further purified, removing odors and ultimately releasing purified and odorless gas.
We innovatively incorporated activated carbon into the filter screen, creating an odor-filtering device that also boasts excellent sealing and leakage prevention capabilities. This feature provides a valuable component for future teams in need of filtering out bacterial liquid odors or other gas odors.
Exhaust Pipe
We designed a unique exhaust pipe, drawing inspiration from Pasteur's gooseneck bottle design (Figure 4). The exhaust tube follows a diagonal downward path, with two bends before it terminates vertically downward. This configuration efficiently directs external bacteria from the air outlet into the odor filter without interrupting the smooth flow of gas discharge. Future teams can consider using this type of pipe as a gas outlet.
Connectors
To ensure a robust and airtight seal within our device, we utilized a chuck with a 5.05 cm diameter as the interface between various components. Additionally, a silicone gasket was strategically positioned between these two chucks to enhance the sealing (Figure 5). The sealing process involved the use of a medium-sized chuck clamp designed to fit the 5.05 cm chuck. This type of connector has good sealing performance, and when sealing devices are needed, this type of connector can be considered.
