This year's project (BCAID) is very creative and challenging. BNUZH-China have tried to build a Bacterial Cellulose(BC) composite scaffold with living cells to assist in the healing of large skin wounds, which is unprecedented in the iGEM competition, and is prospective even in the entire related research field. We work on constructing new gene pathways, developing new parts, and supplementing information for existing parts. We also designed and produced the hardware matching the project, which was produced with 3D printing technology to make the BCAID use process more scientific and convenient.

To prevent the engineered fibroblast ATCC CRL-2522(BJ) from the continuous secretion of cellulase after wound healing, we decided to choose a red light suicide regulatory system to kill the engineered BJ after its mission has been completed. In order to find a light controlled system with better performance, we screened many eukaryotic red light controlled systems (For more details: Engineering), and finally decided to use the red/far-red light system REDMAP¹, developed by Ye Haifeng team of East China Normal University which was recently reported in 2022. This is the first time that REDMAP system has been applied in iGEM competition. The specific mechanism of this system is that when exposed to red light (660 nm), the transactivator (FHY1–VP64) can specifically bind to the light sensor domain (ΔPhyA–Gal4) in the presence of the photosensitive pigment PCB, and the combined protein complex translocates into the nucleus where it can bind to its synthetic promoter (P5 × UAS, 5 × UAS-PhCMVmin) to initiate expression of target genes. Following the exposure to far-red light (730 nm), the transactivator dissociates from the light sensor domain (ΔPhyA–Gal4), thereby terminating the expression of target genes. Compared with existing systems, REDMAP not only has higher efficiency, but also is able to make an activation or inactivate response to changes in light quickly. Furthermore, the REDMAP system is small and can be introduced into cells using either Adeno-associated virus (type 6, AAV6) or lentivirus as the vector to initiate the stable expression of foreign genes.

Our experimental results show that the REDMAP system is very sensitive and that the downstream target gene(MazF) can be efficiently expressed. Future iGEM teams can refer to this system and apply it to biological research that requires high precision and efficient regulation.

In conclusion, the BNUZH-China 2022 team has tried to use an excellent eukaryotic red light controlled system, and we have entered the relevant Parts into the database, which provides the future iGEM team more information to choose from.

Fig.1 Schematic diagram of the basic process of REDMAP system: photoinduced binding (660 nm) and dissociation (730 nm) of PhyA and FHY1.

MazF from E. coli is an endoribonuclease that can specifically cut the ACA sequence of free mRNA. MazF can cleave almost all mRNA, and it can play a toxic role in other kinds of microorganisms to inhibit protein synthesis and cause cell apoptosis, so it is widely used in the genetic modification system of prokaryotes, often used as a suicide gene.

Some projects of iGEM over the years have also used MazF as a suicide gene, but they all used this suicide system in prokaryotic cells. However, no team has applied it to eukaryotic cells. Research shows that the application of MazF as a suicide gene in Pichia pastoris expression system has been relatively mature. Therefore, it is quite possible that MazF can be successfully applied to eukaryotic cells. In fact, the induction of MazF in mammalian cells has been proved to cause programmed cell death. Studies² have shown that adenovirus-mediated delivery of MazF ribonuclease can kill 50%-80% of target cells (HCT116 cells). After being infected with the adenovirus carrying this system, it only takes 72 hours to measure about 35% cell survival, which has great lethality to human cells.

BNUZH-China 2022 team applied MazF to the suicide system of mammalian cells. We use lentivirus as the carrier to transfer the REDMAP system into BJ cells. After the BC scaffold has been degraded, we will use the red light (660nm) to start the synthesis of MazF toxin protein and kill the engineered BJ cells. Our experiments demonstrate that MazF protein can be effectively produced and play its role under the control of red light, which proves the feasible of the suicide system based on MazF in mammalian cells. Our work is a precedent in iGEM competition, which provides experience accumulation and technical summary for later teams. Meanwhile, we have added the obtained data and information to the existing MazF part (BBa_K302033), which is a great contribution to iGEM competition.

We intended to develop a functional circuit board with red, blue, and yellow LED lamps on the circuit board. Different lamps can emit light of specific wavelength to control the light control systems.

Fig.2 Schematic diagram of the LED circuit board

The LED light emitting panel uses MicroUSB to provide 5V power supply. Because all LEDs work at 2-3V, R1-R6 is used for voltage division to make LEDs work at normal voltage. R4-R6 is an adjustable potentiometer, which can fine tune the brightness of LED lamp. U1-U3 (yellow LED) lights up immediately when the power cord is plugged in. When KEY1 is pressed, U4-U6 (blue LED) or U7-U9 (red LED) will be lit. KEY2 will select the part to be lit. U7-U9 (red LED) will be lit when the button is pressed, and U4-U6 (blue LED) will be lit when it pops up.

Fig.3 The PCB drawing of the LED circuit board

Fig.4 The Physical photos of the LED circuit board (already embedded in the shell and in working condition). a. Front b. Back

The basic function of our hardware is to avoid light and prevent the optical control system from being started by mistake. After discussion and brainstorming, we decided to design a cylinder-shaped product to wrap the hurt limbs of patients.

In order to implement and improve our design, we used 3D printing technology to produce our products. 3D printing technology allows us to use various materials to make products, which can be plastic or even metal. When selecting materials, we noticed the new biodegradable material polylactic acid (PLA). PLA has the advantages of good biocompatibility, safety, environmental friendliness and low price³. Therefore, we decided to use PLA to make the shell. The use of degradable and renewable materials conforms to the concept of environmental protection and sustainability of iGEM. Meanwhile, PLA is also a light material, which will not bring too much burden to patients.

We modeled the shell on the computer and used 3D printing technology to make the shell. Firstly, Soildworks is used for modeling. Two semi cylindrical shells were designed to enclose the arm (Fig.5). After the model was completed, the 3D printing process of FDM is adopted. We used PLA to produce the shell, which is environmentally friendly. Meanwhile, we used black PLA to avoid light. Embedded nuts were placed onto the two semi cylindrical shells by heating (Fig.7a). The two semi cylindrical shells were then connected with tiny hinges. The shell can be quickly assembled and disassembled with Velcro (Fig.7b).

Fig.5 Schematic of modeling with Soildworks

Fig.6 The scene of 3D printing

Fig.7 a. Nut and hinge b. The quick assembling with Velcro

The function of hardware is not only to avoid light, but also to cooperate with the treatment process. In order to conveniently observe the state of the engineered cells to understand the treatment progress and adjust the treatment plan, we installed a small camera on the hardware (Fig.8). In order to precisely control the light control systems, we installed a PCB circuit board on the hardware. There are red, blue, and yellow LED lights on the PCB function board. Different lights can emit light of specific wavelength to activate the corresponding the light control system. After discussion and research, we made two holes on the cylindrical shell. So, the camera and LED circuit board can be placed on the upper part of the shell through screws (Fig.9a). In addition, the sleeves are used to furtherly block the light (Fig.9b). Our design can achieve a light free environment inside the hardware.

Fig.8 We added camera and PCB.

Fig.9 Some representative parts of our hardware. a. The camera and PCB function board are fixed on the shell b. The sleeve is light shielded to achieve a light free environment

Based on the digital documents needed for 3D printing and manufacturing hardware, four views of parts are derived and displayed (Fig.10), which can help other engineers understand our products better and make our product design more standardized.

Fig.10 Four views of parts

Over the years, the use of 3D printing technology in iGEM is relatively rare. 3D printing technology is a rapid prototyping technology that can provide users with highly personalized physical products with short production cycle. Notably, iGEM participants often have a high demand for personalized customization when making physical objects. In addition, it is quite common for them to produce products in a short time. We believe that the characteristics and advantages of 3D printing technology are in line with the needs of iGEM competition participants. We hope that our work on 3D printing technology can enrich iGEM's content and provide useful experience and reference for other teams.

1 Zhou, Y. et al. A small and highly sensitive red/far-red optogenetic switch for applications in mammals. Nat Biotechnol 40, 262-272 (2022).

2 Shapira, S. et al. Selective eradication of cancer cells by delivery of adenovirus-based toxins. Oncotarget 8, 38581-38591 (2017).

3 Fang, X., Chen, S. & Wu, Z. Research progress of modification and application of polylactic acid. Speciality Petrochemicals 28, 71-76 (2011).