Summary

In the sixth year of our team participated in the iGEM competition, we decided to solve the problems in space exploration. Our team considered response from human practice throughout. Therefore, we come up with to four aspects to tackle martian immigrants.

Yeast and food production

Synthesis Lycopene, nerol, limonene using engineered yeast and optimized production

Yeast can play an important role in space food production, especially in providing sustainable nutrition for astronauts on long-term missions. With some engineering, yeast S. cerevisiae can be further bolstered to add nutrients, taste, color, and texture to the meal, by synthesis food pigment or essence. Therefore, we choose yeast strain CEN.PK2-1C as a chassis. It's safe and widely used. We hope to synthesize lycopene, nerol and limonene inside its' cell.

Based on our design, we selected three key enzymes- crtE, crtI and crtY to synthesize lycopene. Meanwhile, we introduced p-protein P2A and procine IGG6 to form a polycistrine substructure to express these three enzymes simultaneously. To transform the gene circuit into the yeast, a yeast shuttle plasmid was selected, whose replication initiation site in yeast was 2 micro and screening marker was URA3 (Figure 1A) . We ordered the gene from our sponsor, and sequence with sanger method after assembly (Figure 1B). After that, We transformed the correct plasmid into Yeast CEN.PK2-1C (a common strain with promising safety), the colony was selected for successful transformants using SD-URA3 nutrient deficit medium, and used colony PCR as double-check (Figure 1C). Then, we selected the correct colony for fermentation.

We extracted lycopene with ethyl acetate, and tested it with spectrophotometer. Meanwhile, we carefully checked whether the medium had color changes (lycopene showed yellow color).

From the first round of experiment, we realized that the amount of lycopene we synthesised was low, therefore increasing the yield become our next goal.

By doing literature research, we found a system called HapAmp that can integrate multiple copies in yeast. We purchased the plasmid contains the part we want from addgene(pIT6EG7ml, #185886), which has no replication site for integrating into genome. The screening label for it is Leu2 (Figure 2A). After the plasmid is confirmed by sanger sequencing (Figure 2B,C) , we extract it and transform it to Yeast CEN.PK2-1C (Figure 2D).

After fermentating, we extract lycopene with ethyl acetate and then we test it with a light spectrophotometer The increased A474 suggested the success of our design(Figure 2E). At the same time, we also carefully checked the medium for color changes (lycopene appears yellow), the product can be visualize by the colour of culture.

In our Human practice session, the psychologist we interviewed reminded us that we need to take into account the emotional changes of the people in the complex environment of Mars. We noticed that nerol and limonene can play a role in this, therefore we used the GmNES and limonene synthesis module based on the HapAmp system (Figure 3A,B, Figure 5A,B).

After confirming that the plasmid was correct by sanger sequencing (Figure 3C, Figure 5C), we extracted them and transferred them to Yeast CEN.PK2-1C respectively. Correct colonies were confirmed by PCR (Figure 3D, Figure 5D).

After expression, we extracted the product with ethyl acetate and used spectrophotometry (Figure 3 E,F, Figure 5E,F) to measure A252. And using Gas chromatography/mass spectrometry using (GC/MS, flow phase ethyl acetate) to confirm the presence of the product. (Figure 4, Figure 6).

The result of GC-MS also suggested limonene were synthesized.

The result of GC-MS also suggested nerol were synthesized.

Therefore, the GC/MS result confirmed that the production of limonene and nerol was successfully produced.

Engineer cyanobacteria

Generating carbon source from the Sun

Cyanobacteria can use carbon dioxide in Mars and release oxygen, at the same time, produce sucrose, and utilize as carbon source for our engineered yeast and our potential chassis. These make it a promising chassis for Martian migration.

However, algae are greedy just like Mr Plankton, the sucrose produced by cyanobacteria can not be export to the outer cell. and second is that iGEMers do not dare to use it as chassis due to limited conditions of experiment. So we want to engineer cyanobacteria to export sucrose with high titer, and also to give a more friendly setup to engineer cyanobacteria. We choose Synechococcus elongatus 7942, a safe and widely used chassis.

In all cyanobacteria-based co-culture systems, enabling cyanobacteria to secrete sucrose is very important, so our first step is to express CscB in cyanobacteria (Figure 7A,B), which is a sucrose efflux protein from Escherichia coli. After constructing and confirming the correctness of the plasmid (Figure 7C), we transferred the plasmid into 7942. After checking by PCR (Figure 7D), we cultured cyanobacteria for 7 days, and finally tested whether there was sucrose excretion. By treating supernatant with resorcinol, we can quantify sucrose by spectrophotometry (Figure 7E). Our engineered strain successfully exported sucrose (Figure 7F).

In the last round of DBTL, we succeeded in giving cyanobacteria the ability to export sucrose and we hope to increase the amount of sucrose secreted, which will provide more possibilities for the co-culture system.

We introduced SPS and GlgC, two enzymes that are important in the formation of sucrose. After constructing and confirming the construction of the plasmid (Figure 8A,B,C), we transferred the plasmid into 7942, and confirmed the transformers by PCR (Figure 8D). Then we selected cyanobacteria for culture for 7 days, and finally tested whether secreted sucrose. We also simultaneously cultured strains expressing only CscB as controls to confirm the functions of SPS and GlgC. This design worked better than last (Figure 8E).

In the Human practice session, after discussing with others iGEMer during in Space meating, we realized that encorage other iGEMers to try to solve the Mars migration problem could achieve better results than we could do it alone.However, many iGEMer believes that cyanobacteria require additional culture equipment and the operation cycle is too long to be used in iGEM competitions. So after completing two DBTL cycles, we hope to improve the growth rate of cyanobacteria and provide a more friendly culture method.

After reviewing the literature, we constructed the plasmid expressing katG and transferred it into Polycoccus 7942 (Figure 9A,B,C). After confirmed by PCR(Figure 9D), we cultured it with wildtype under fluorescent lamps (Figure 9E,G), and found that the engineered bacteria had a faster growth rate and could reach the level of transformation in less than 4 days (Figure 9F).

Screen resistant Azotobacteria and directed evolution

While producing food in a fermentor using engineered yeast is a potential solution, traditional soil-grown food better meets human mental needs. However, the soil of Mars is too poor to plant and contains perchlorate(0.4 to 0.6 wt%), a toxic component to plants to growth. Based on our test, 0.5% perchlorate salt will inhibit the plant growth.Therefore, we decided to engineer chlorite resistance azotobacteria, which can fix nitrogen for plants and also survive on mars.

We first tried to screen resisted azotobacteria from commercial safety-promising nitragin. Then, we demonstrated that directed evolution can be used to increase bacteria's resistance to chlorite. Finally, we build a hardware for continous evolution (Hardware)

Although yeast can produce food for us in Mars, it's not a long-term solution. We need plant. However, the soil of Mars is too poor to plant. So we decided to engineer azotobacteria, which can fix nitrogen into ammonium for plant. While toxic chlorite in the soil will kill azotobacteria, we tried to screen resisted azotobacteria from commercial safety-promising nitragin. Then, we demonstrated that directed evolution can be used to increase bacteria's resistance to chlorite. Finally, we build a hardware for continous evolution (Hardware).

After trial and Human practice, we hope to isolate nitrogen-fixing bacteria that are tolerant to chlorite. We use the commercial rhizobium agent provided by Jian Huang, which is a fertilizer containing nitrogen-fixing bacteria and has promising safety We screened the strains on Ashuria media supplemented with chlorite and amplified the clones by PCR (Figure 8 A,B). After sequencing, blast (blast.ncbi.nlm.nih.gov) was used to check the species of the strains. Then we found it's Pseudoxanthomonas mexicana, a reported azotobacteria (Figure 8C).

Directed evolution is a method that can improve the characteristics of strains. We hope to improve the tolerance of bacteria to chlorite by directed evolution. We selected Escherichia coli DH5α as the test strain and continuously generated mutations on its genome through MP6. By increasing chlorite concentration in the medium, the beneficial mutation will be enriched, and finally achieve the purpose of evolution. We transfer MP6 into DH5α, and then start the directed evolution after PCR confirmation.

We gradually increased the concentration of chlorite in LB medium and measured A600 during the directed evolution experiment (Figure 9B, C). After two weeks, we compared the evoloved strain vesus wildtype by inoculated them into LB with 1mM chlorite. We can see evolved E. coli grew better than wild-type.

Accerate the DBTL of plant synbio

Permanent genomic insertion capabilities for Carbon nanodot-based tracked, transformation, translation, and trans-regulation (TTTT) system

Plant is crucial for Mars, and plant synthetic biology can make difference. But as the heart of synthetic biology, iGEM is considered too time-pressed to choose plant as chassis. So here, we want to accelerate the DBTL cycle from two aspects. The first is permanent genomic insertion capabilities for Carbon nanodot-based tracked, transformation, translation, and trans-regulation (TTTT) system.

Genetic engineering of plants is a crucial technology for both basic research and agricultural applications. Carboxyl-modified carbon dots (CDP) with a positive charge can serve as plant transfection vectors. Their small size (3-5 nm) allows them to pass through cell biofilms more easily than carbon nanotubes.

However, in year 2023, our TTTT system can only preform transient expression through leaf spraying. This drawback limits the application scenarios of it. This year,bBy enhancing the preparation process of CDP, the latest version, known as Smart-CD (patent pending), can effectively transfect plants through leaf spray for transient expression and seed soaking for genomic insertion since the amount of DNA delivered was enough to preform homology-directed repair(HDR). This method has been tested on and proven to work on several common crops.

In our experiment outside the iGEM project, 9 types of vegetables traits were tested following the seed transformation and was transformed into a vector containing firefly luciferase. The plant was then cultured in soil medium under normal condition until grown larger.

As part of our iGEM project, our team collaborated with the Huang Lab to conduct experiments validating protein expression in two plant models: Sorghum bicolor, a common energy crop, and Arabidopsis thaliana, after seed soaking. The transformed plants were sprayed with a D-Luciferin, Potassium Salt solution (10 mg/mL in D-PBS) and immediately imaged using the CCD camera of the Chemi Blot System. Both Arabidopsis and Sorghum exhibited strong luciferase expression, indicating successful transformation. Notably, the luciferase signal was also detected in the seed pods, suggesting that the genetic modifications may be inheritable.

The protein expression was also verified by detecting the GFP tag using western blot. In our experiment, 100mg arabidopsis leaf was crushed after being frozen by liquid nitrogen, 250ul RIPA lysis buffer(Thermofisher 89901), 1ul Pierce™ Universal Nuclease(Thermofisher 88700) and 2.5ul Halt™ Protease Inhibitor Cocktail, EDTA-Free(Thermofisher 87785) was added to the tissue and incubated at 4℃ for 1hr. 30ul of the extracted protein was added with 10ul 4X SDS-sampling buffer and heated at 95℃ for 5min and loaded 30ul to the gel. The gel was then goes to semi-dry transfer to a PVDF membrane and be blot with 1:5000 dilution of primary antibody: Rabbit anti GFP-Tag mAb (Abclonal, AE078) and Actin(Plant Specific) Rabbit mAb (Abclonal, A23959) at room temperature for 2 hours and detected by Pierce™ Fast Western Blot Kit, ECL Substrate(Thermofisher, 35050).

The western blot result shown that the GFP was successfully expressed.

Migrate spiritual civilization by DNA

Besides material conditions, spiritual civilization is also very important. In Model United Nations held by us, we noticed storage of our culture in Earth got a lot of enthusiasm, so we wanted to migrate spiritual civilization by DNA storage.

The system was now avaliable on the terminal of our wiki at the right, use /DNA storage command to try it. More story can also be found in the file storage of the terminal.