Team:BUCT-China/Implementation

PHFA
CulturedMeat

Implemention

In our project, we get two products available for the real world. One is PHFA, the raw material of scaffolds for cell growth. The second is our final product, cultured meat. PHFA not only has a certain supporting strength, but also has broad application prospects in biodegradable tissue engineering materials. Using tissue engineering knowledge, PHFA was used as the main biological scaffold material to provide growth sites for chicken derived muscle satellite cells. Finally, 3D printing technology was used to construct cultured meat.

Part1.Polyhydroxy fatty acid (PHFA)

End users：

Our scaffold raw material PHFA can not only be used to produce cultured meat, but also be used as a degradable biomaterial. PHFA can be used as a scaffold material for 3D printing technology, so any 3D printing-related company and its products can become our end users. In addition, PHFA can be also used to make degradable plastic bottles and bags. As environmental protection becomes a global issue, any country and region could be our potential end user, thereby alleviating environmental pollution.

Envision：

In the future, with more in-depth research and development of PHFA, PHFA can be mass-produced on an industrial scale. Therefore, PHFA will become a more mature scaffold material and provide a better culture environment for cultured meat. What’s more, PHFA will also replace the existing plastics with polyethylene as raw material in all aspects. It will be the answer for environmental protection problem.

Implementation：

In this project, we mainly focus on PHFA as a scaffold material for 3D printing technology for real-world use. The printed scaffolds are used for the growth and differentiation of muscle and liver cells. PHFA is mixed with collagen in an appropriate proportion to form a bioink. The mixed bioink encapsulates muscle satellite cells, prints them onto a culture dish, and cures them with a curing agent to maintain the shape of the deposit. The bioink encapsulation realizes the directional growth of the cells, and the degradation of the bioink forms pore canals to enable the cells to obtain an internal proliferation channel and migrate to the surface of the bioink through the degradation pore canals so as to realize highdensity 3D cell culture. High density of muscle satellite cells is beneficial to the formation of myotubes.

In addition, we have also designed a set of feasible processes for the industrial production of PHFA. The specific production process is shown in the figure below:

Firstly, the transformed E. coli was inoculated in the small fermentation tank (small jar) for fermentation and culture as seed liquid. Then it was transferred to a large fermentation tank (big jar), expanded culture and accumulated to produce the target product PHFA.

When the amount of PHFA is accumulated enough, the fermentation liquid is transferred to the next device for cell wall breaking, and the cell wall and cell membrane are destroyed to a certain extent by physical crushing and high-pressure homogenizer, so as to maximize the release of the intracellular product PHFA into the liquid phase.

Solid-liquid separation is achieved by extraction technology. Solid suspended solids, such as thallus and cell fragments, were separated and removed to obtain pure PHFA. Trichloromethane is added to the fermentation liquid mixture, and PHFA is separated according to the solubility of the fermentation liquid components in the aqueous phase and the organic phase.

In order to further obtain PHFA pure products, ion exchange resin was used to purify PHFA from fermentation broth. Ion exchange resin is a polymer compound with acid or alkaline function, so it can exchange anion and cation. The fermentation product PHFA has acidic functional groups, which can exist in the ionic state in solution and exchange with the active functional groups of the resin.

Beyond the engineering process of producing PHFA, our partner SCAU-China provides us a system of suicide genes that are intrigued by near-infrared light, which is theoretical plausible to be used in our chassis bacteria. This suicide system will generate artificial designed nucleus to digest euchromatin and lead dead cell eventually to secure biosafety needs in the future. We are greatly appreciated SCAU-China help us to take bio-safety in consideration and provide technical support about this system in the future.
https://2021.igem.org/Team:SCAU-China/Safety

Safety Concern：

Using the designed system won’t cause damage to nature and humans directly, but it makes the genes of fadL and P450 BM3 invading the gene pool of E. coli. If the E. coli inside the system are leaked out, it may influence the ecosystem in unpredictable way. It may just act as artificial accelerating evolution of these bacteria by having a better fatty acid absorbing efficiency compare to natural species, or it may cause fatty acid material unreliable under natural condition due to the existence of novel bacterium.

Future challenges：

In accordance with all above, we don’t what to restrict our imagination by the current experiments and knowledge. However, we also are self-awareness towards what we need to prove and what challenges that we are going to face. Here, we list three main points. The most important when we want to industrialize our technique is efficiency, which is determined by the efficiency of the genes, fadL&P450 BM3, we used, the cost of the whole producing process, the income of Polyhydroxy fatty acid (PHFA) product. We still need more experiments to support or improve the transportation efficiency of fadL and hydroxylation efficiency of the P450 BM3. Besides, more data should be collected and more communication with experts should be carried out to determine more appropriate direct, pore size, material mixing ratio and other related variables to make the most suitable scaffold for artificial meat culture conditions. Last but not least is how to use the PHFA synthesized by E. coli for more diverse commercial uses. These are the key issues of our project, and we will think deeply and try to solve them in the future development.

Part2. Cultured Meat

End users：

We want our cultured meat products to be available to people around the world who need protein. Artificial meat from chicken eggs is not restricted by religion and can provide higher nutritional content. It could give more people on the planet access to the protein they need to survive and make those who eat it healthier.

Envision：

In the future, as people become more aware of environmental protection and pay more attention to their own health, most people will choose to eat cultured meat. Some of them may also enjoy the pleasure of custom-made cultured meat, with its ingredients altered according to people's health conditions. For example, obese people can eat modified artificial meat with lower fat content.

Implementation：

For the promotion and publicity of cultured meat products, our team has two plans. In terms of knowledge popularization, we should spread the idea that cultured meat is healthier and more environmentally friendly to the public, so that more people can accept cultured meat psychologically. On the product side, we still need to further improve the cultured meat products, evaluate it in terms of taste, so that people can accept it physically.

Safety Concern：

The development of cultured meat involves many disciplines, the technology of discipline integration is not mature, the safety evaluation system has not been completely established, and there are still some safety unknowns in the production process, such as the serum components of natural culture medium, whether the primary cells will have mutations in the extraction and culture process, etc. These are all security issues worth thinking about.

Future challenges：

How to better reduce the cost and expand the output, the experiment from the laboratory to the industrial application of the factory, and what kind of cultured meat products should be developed to meet the needs of the market are all the challenges we need to take into account.

Reference

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