iGEM 2024 — Team sz-shd

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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# Notebook

![](https://static.igem.wiki/teams/5308/icons/team-logo.png)
SZ-SHD

* [Previous page](%7B%7B%20url_for%28%27home%27%29%20%7D%7D)

# Notebook

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block page\_content %}

# Engineering

![](https://static.igem.wiki/teams/5308/icons/team-logo.png)
SZ-SHD

* [Previous page](%7B%7B%20url_for%28%27home%27%29%20%7D%7D)
   [Summary](#Summary)
   [Yeast and food production](#Yeast and food production)
   [Engineer cyanobacteria](#Engineer cyanobacteria)
  [Screen resistant Azotobacteria and directed evolution](#Screen resistant Azotobacteria and directed evolution)
  [Accerate the DBTL of plant synbio](#Accerate the DBTL of plant synbio)
  [Migrate spiritual civilization by DNA](#Migrate spiritual civilization by DNA)

# Engineering success

---

## 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.

![](https://static.igem.wiki/teams/5308/images/engineering/engineering1.png)

Overview of SZ-SHD 2024 project

## 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.

![](https://static.igem.wiki/teams/5308/images/engineering/engineering2.png)

DBTL cycle of engineered yeast

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).

![](https://static.igem.wiki/teams/5308/images/engineering/engineering3.png)

Figure 1. A. The plasmid map and expression cassette. B. Sanger sequencing result of plamid construction. C. Colony PCR of CEN.PK2-1C transformed with plasmid. D. The standard curve of lycopene, A474 was measured by spectrophotometry. E. Culture after fermentation. F. A474 of extraction. Cluster+ means the strain with CrtE-CrtB-CrtI cassette.

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.

![](https://static.igem.wiki/teams/5308/images/engineering/engineering4.png)

Figure 2. A. The plasmid map of lycopene synthesis cassette in HapAmp. B. lycopene synthesis cassette. C. Sanger sequencing result of plamid construction. D. Colony PCR of CEN.PK2-1C transformed with plasmid. D. Culture after fermentation. E. A474 of extraction. Cluster+ means the strain with CrtE-CrtB-CrtI cassette, HapAmp+ means the strain with HapAmp-based cassette.

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).

![](https://static.igem.wiki/teams/5308/images/engineering/engineering5.png)

Figure 3. A. The plasmid map of limonene synthesis cassette in HapAmp. B. limonene synthesis cassette. C. Sanger sequencing result of plamid construction. D. Colony PCR of CEN.PK2-1C transformed with plasmid. E. The standard curve of limonene, A252 was measured by spectrophotometry. F. Cluster+ means the strain with CrtE-CrtB-CrtI cassette, HapAmp+ means the strain with HapAmp-based cassette.

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

![](https://static.igem.wiki/teams/5308/images/engineering/engineering6.png)

Figure 4. Verify production of limonene by engineered yeast

![](https://static.igem.wiki/teams/5308/images/engineering/engineering7.png)

Figure 5. A. The plasmid map of nerol synthesis cassette in HapAmp. B. nerol synthesis cassette. C. Sanger sequencing result of plamid construction. D. Colony PCR of CEN.PK2-1C transformed with plasmid. D. Culture after fermentation. E. The standard curve of nerol, A200 was measured by spectrophotometry. F. Cluster+ means the strain with CrtE-CrtB-CrtI cassette, HapAmp+ means the strain with HapAmp-based cassette.

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

![](https://static.igem.wiki/teams/5308/images/engineering/engineering8.png)

Figure 6. Verify production of nerol by engineered yeast

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.

![](https://static.igem.wiki/teams/5308/images/engineering/engineering9.png)

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).

![](https://static.igem.wiki/teams/5308/images/engineering/engineering10.png)

Figure 7. A. The plasmid expression CscB induced by IPTG. B. Cassette for expression of CscB. C. Sanger sequencing result of plamid construction. D. Colony PCR of 7942 transformed with plasmid. E. The standard curve of sucrose, A480 was measured by spectrophotometry after being treated by resorcinol. F. CscB+ means the strain with CscB expression induced by IPTG, WT means the strain without engineering.

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).

![](https://static.igem.wiki/teams/5308/images/engineering/engineering11.png)

Figure 8. A. The plasmid map of IPTG-inducible CscB-SPS-GlgC cassette. B. CscB-SPS-GlgC cassette controlled by lacI. C. Sanger sequencing result of plamid construction. D. Colony PCR of 7942 transformed with plasmid. D. Culture after fermentation. E. A474 of extraction. CSCB+ means the strain with CSCB cassette, 3 enzyme+ means the strain with CscB-SPS-GlgC cassette.

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).

![](https://static.igem.wiki/teams/5308/images/engineering/engineering12.png)

Figure 9. A. The map of katG expressing plasmid. B. IPTG-inducible KatG expression cassette. C. Sanger sequencing result of plamid construction. D. Colony PCR of 7942 transformed with plasmid. E. The growth curve of 7942 cultured under different conditions. F. The growth curve of engineered strain and wildtype. They were cultured under a light lamp. G. Picture of cultured cyanobacteria.

## 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.

![](https://static.igem.wiki/teams/5308/images/engineering/new.png)

Toxicity of chlorite salt to plant under 0.5; 1.0wt%

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](https://2024.igem.wiki/sz-shd/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](https://2024.igem.wiki/sz-shd/hardware)).

![](https://static.igem.wiki/teams/5308/images/engineering/engineering13.png)

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](https://blast.ncbi.nlm.nih.gov/Blast.cgi)) was used to check the species of the strains. Then we found it's *Pseudoxanthomonas mexicana*, a reported azotobacteria (Figure 8C).

![](https://static.igem.wiki/teams/5308/images/engineering/engineering14.png)

Figure 10. A. Screen of chlorite-tolerated azotobacteria from commercial rhizobium agent. B. Amplify 16S DNA. C. Check isolated azotobacteria.

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.

![](https://static.igem.wiki/teams/5308/images/engineering/engineering15.png)

Figure 11. A. Plasmid map of MP6 we used. B. The change of A600 (OD) during directed evolution. The experiment setup and be checked in C. D. Comparision of wildtype E. coli (right) and evolved E. coli (left), 1 μl was inoculated into LB with 1mM chlorite and cultured ovenight.

## 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.

![](https://static.igem.wiki/teams/5308/images/engineering/engineering16.png)

![](https://static.igem.wiki/teams/5308/images/engineering/engineering17.png)

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.

![](https://static.igem.wiki/teams/5308/images/engineering/new2.png)

Overview of expanded TTTT system's application scenarios

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.

![](https://static.igem.wiki/teams/5308/images/engineering/engineering18.png)

Transient/ Genomic insertion vector expressing firefly luciferase

![](https://static.igem.wiki/teams/5308/images/engineering/engineering19.png)

Expression of firefly luciferase in different vegetable traits after seed soaking

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.

![](https://static.igem.wiki/teams/5308/images/engineering/engineering20.png)

a. Sorghum plant grown after seed soaking(by Huang lab); b. Sorghum leaf expressing Firefly luciferase 24days after seed soaking c. Arabidopsis leaf expressing Firefly luciferase; c. Arabidopsis seed pod and seeds expressing firefly luciferase(more info at engineering success)

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).

![](https://static.igem.wiki/teams/5308/images/engineering/new3.png)

Western blot result of TTTT seed soaking arabidopsis

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.

![](https://static.igem.wiki/teams/5308/images/engineering/engineering21.png)

Work flow of DNA encoding system

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.

![](https://static.igem.wiki/teams/5308/images/engineering/new4.png)

Work flow of DNA encoding system

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# Experiments

![](https://static.igem.wiki/teams/5308/icons/team-logo.png)
SZ-SHD

* [Previous page](%7B%7B%20url_for%28%27home%27%29%20%7D%7D)
  [Protocols](#protocols)

# Experiments

## Protocols

### PCR :

1. Extract 2 ul of sample/DNA, mix it with 2ul of reverse primer, 2ul of forward primer, 25ul of Phanta Flash Master Mix and 19ul of dd h3O as buffer in a clean, sterile tube.
2. Place the tube containing the reaction mixture in the thermal cycler and start the program. The thermal cycler will cycle through denaturation, annealing, and extension steps multiple times. The number of cycles depends on the starting amount of DNA and the desired level of amplification.
3. After the PCR reaction is complete，using gel electrophoresis to visualize the amplified DNA fragments and determine their size.

### Gel preparation:

1. Put 0.4g agarose into a conical flask and mix it with 40mL TAE solution. Then, microwave the mixture for 1 minute.
2. Add 4ul nucleic acid dye into the mixture and shake the mixture gently.
3. Finally, decant the gel into adhesive board and insert a gel comb with appropriate number of holes.
4. Wait for around 10 minutes.

### Agarose Gel Electrophoresis :

1. Add 1/10 of sample loading buffer into the sample and put the gel into the electrophoresis tank. Then, add sufficient buffer which could cover the gel.
2. Then, add the sample into the gel with moderate amount of DNA marker. Wait 30 minutes to let the DNA run out.

### Cutting Gel:

1. Visualize the gel under UV light in a gel documentation system . The DNA bands will appear as fluorescent spots.
2. Then put the gel in a handheld UV lamp. Put on appropriate protective gear such as gloves and safety glasses to protect against UV radiation.
3. Using a clean scalpel or a gel cutting tool, carefully cut out the desired band of DNA. Try to make clean cuts to minimize contamination and damage to the DNA. And put the gel with DNA into centrifuge tubes separately.

### Gel extraction:

1. Add three times the volume of the gel of PE solution to the gel cut off seperately and melt the gel in room temperature for around 10 minutes.
2. Transfer the solution obtained into adsorption column CA5 and place the columns for 2 minutes in room temperature. Then centrifuge the solutions at a speed of 12000rpm for 60 seconds. Then pour out the waste in the collection tubes.
3. Add 600 ul of Buffer-PW into adsorption column CA5 and centrifuge the solutions at a speed of 12000rpm for 60 seconds. Then pour out the waste in the collection tubes and put the adsorption column CA5 into the collection tubes.
4. Repeated step 3. Put the adsorption column CA5 into the collection tubes to centrifuge the solutions at a speed of 12000rpm for 60 secongs until no Buffer-PW remains.
5. Place the adsorption column CA5 in room temperature for few mintues until the adsorption column CA5 are completely dry.
6. Put the adsorption column CA5 into clean centrifuge tubes and add 30 ul Elution buffer TB and place the solution in room temperature for 2 minutes. Then centrifuge the solution for 2 minutes at a speed of 12000 rpm.
7. Examine the purity of the DNA extracted.

### Gibson assembly

1. Replicate the DNA fragment that required to be assembled by PCR.
2. Cut the 5’ end of the DNA fragment.
3. Stack the single-stranded DNA fragments together and assemble them by base pairing.
4. Fixing the unpaired bases by DNA polymerase and DNA ligase.
5. Transfer the assembled DNA fragment into the host cell.

### Transformation

1. Mix the chemically competent cell with plasmid DNA.
2. Incubate the cells with DNA on ice for 5‒30 minutes.
3. Perform heat shock at 37‒42°C for 25‒45 seconds as appropriate for the bacterial strain and DNA used.
4. Return the heat-shocked cells to the ice for more than two minutes.

### Bacteria culturing

1. Prepare the medium with appropriate antibiotic.
2. Prepare the cell suspension that required to be cultured or screened.
3. Spread the cell suspension with a cell spreader.
4. Incubate the plate in an incubator at an appropriate temperature.

### Bacterial colony PCR

1. Mix 2ul of reverse primer, 2ul of forward primer, 25ul of Phanta Flash Master Mix and 21ul of dd h3O as buffer in a clean, sterile tube.
2. Choose few single colonies and mark them on the plates.
3. Add the colonies to the PCR system.
4. Place the tube containing the reaction mixture in the thermal cycler and start the program. The thermal cycler will cycle through denaturation, annealing, and extension steps multiple times. The number of cycles depends on the starting amount of DNA and the desired level of amplification.
5. After the PCR reaction is complete，using gel electrophoresis to visualize the amplified DNA fragments and determine their size.

### Liquid culture of bacteria

1. Prepare liquid LB medium with appropriate antibiotics.
2. Choose single colony and add to the the LB medium with the tip of pipette.
3. Blend the colony with LB medium by pipette tip.
4. Cover the culturing container with bacteria-free aluminum foil or a non-hermetic cover.
5. Culture the bacteria in a shaking incubator at an appropriate temperature.

### Plasmid extraction

1. Cultivate bacterial samples overnight in growth medium with antibiotic selective pressure.
2. The bacterial culture is centrifuged and the supernatant is discarded.
3. A buffer is added to the tube containing bacterial pellet in order to resuspend the pellet.
4. Add the lysis buffer and gently invert the tube multiple times to fully lyse the cells.
5. Neutralization buffer (typically potassium acetate solution) is added and gently inverted to neutralize the alkaline conditions and lower the pH.
6. The mixture is then centrifuged, where genomic DNA and precipitated proteins form a pellet and the plasmid DNA remains in the supernatant.
7. Add 100% ethanol to the supernatant in order to precipitate plasmid DNA. Once precipitated, the precipitate containing the plasmid DNA is rinsed in 70% ethanol to remove any remaining salts and impurities.
8. Leave the tube open to remove all the residue alcohol by evaporation.
9. Resuspend the DNA pellet in a buffer solution containing Tris and EDTA.
10. Pass washing buffer through the column while the DNA remains bound to the silica.
11. Repeat the washing step to remove endonucleases, RNA, proteins, dyes, and low-molecular weight impurities.
12. Elute the plasmid DNA with sterile water or an elution buffer.

### Yeast culture

1. Inoculate yeast into medium under sterile conditions. The inoculation can be performed using tools such as an inoculation ring, an inoculation needle, or a pipet.
2. Single colonies of yeast recipient bacteria were inoculated on YPD plates and cultured for 2 days at 30 ° C.

### Preparation for competent cell of yeast

1. Single colonies from the plates were picked and inoculated in 10ml YPD liquid medium and shaken overnight at 30°C on a shaker.
2. After overnight culture, the cells were inoculated into 100ml YPD medium at about 1% inoculum and cultured by shaking to an OD value of about 1.2.
3. The precipitated bacteria were collected by centrifugation at 5000 rpm for 5min at 4°C and resuspended in 100ml of precooled sterile water.
4. The precipitated bacteria were collected again by centrifugation at 5000rpm for 10min at 4°C and resuspended in 100ml of precooled sterile water
5. The cells were washed with 20ml, 1mol/L sorbitol.
6. The cells were dissolved in 200μl, 1mol/L pre-cooled sorbitol without glycerol and placed at -80°C for several hours until transformation.

### Transformation of Yeast

1. About 100ng DNA (plasmid) was added to the competent cells, and the mixed solution was aspirated by pipettor.
2. PEG at 600μl was added and mixed at high speed (10S-1 min).
3. Recovery was performed at 30°C with shaking for 30min.
4. 70μl DMSO was added to the ultra-clean table (under sterile conditions), and the mixture was gently reversed.
5. Heat shock in 42°C for 15min.
6. Leave on ice for 1-2min.
7. The samples were centrifuged at 14000rpm for 15s and the supernatant was discarded.
8. Cells were resuspended in 300μl of 1×TE.
9. 100μl were plated on TPDA plates and incubated at 30°C for 48 to 72h to observe the transformed colonies.

### Bacterial PCR for Yeast

1. Mix 2ul of reverse primer, 2ul of forward primer, 25ul of Phanta Flash Master Mix and 21ul of dd h3O as buffer in a clean, sterile tube.
2. Choose few single colonies and mark them on the plates.
3. Add M5 Hiper Super Speed Mix directly amplifies the best companion to the colonies in a tube.
4. Centrifuge the colonies with M5 Hiper Super Speed Mix transiently and use 1-2ul as PCR template.
5. Place the tube containing the reaction mixture in the thermal cycler and start the program. The thermal cycler will cycle through denaturation, annealing, and extension steps multiple times. The number of cycles depends on the starting amount of DNA and the desired level of amplification.
6. After the PCR reaction is complete，using gel electrophoresis to visualize the amplified DNA fragments and determine their size.

### Inoculation

1. Add 90ul bacteria-free liquid (dd h3O or liquid LB medium without antibiotics) into 8 PCR tubes separately.
2. Add 10ul culture material of bacteria to the first PCR tube and mix it with dd h3O with a pipette.
3. Obtain 10ul of the cell suspension from the first tube and add it to the second PCR tube. Then mix the liquid with pipette.
4. Continuing the serial dilution and the concentration of the cell suspension in the last (8th) tube is 10^(-8).
5. Obtain 1.5ul of the cell suspension from each tube and inoculate on the agar LB plate with appropriate antibiotics.

### Coercive evolution

1. 1. Add 100uM sodium chlorite into 3ml liquid LB medium.
2. 2. Add ampicillin antibiotics and 50ul previous E.coli bacteria solution.
3. 3. Add 3ul 1ug/ml Anhydrotetracycline Hydrochloride(aTc) for inducement.
4. 4. Culture overnight under appropriate conditions.

### Culture of Cyanobacteria

1. Prepare 20ml 1.7g/L BG11 medium with 100mM sodium bicarbonate (by mixing 0.16802g sodium bicarbonate and 0.034g BG11 medium with 20ml water).
2. Culture 3ul Cyanobacteria sample in 5ml BG11 medium within a centrifuge tube for few days under the light source, such as sunlight or light box.
3. Examine the OD600 of cyanobacteria every day.

### Screening of nitrogen fixing bacteria

1. Obtain 1g of root nodule or soil sample and add to 20ml dd h3O within a 50ml centrifuge tube. Grind the sample if sample is Obtain 1g of root nodule or soil sample and add to 20ml dd h3O within a 50ml centrifuge tube. Grind the sample if the sample is a root nodule.
2. Shake the cell suspension and stew for 10 minutes.
3. Dilute the cell suspension 1000 times.
4. Spread 40ul 1x, 100x, 1000x cell suspension on the Ashby Mannitol Agar Medium plates with sodium chlorite separately.
5. Culture overnight at 30°C.

### Extraction of plant tissue protein:

1. Weight 100mg arabidopsis leaf in a 2ml EP tube, crushed after freeze by liquid nitrogen.
2. Add 250ul RIPA lysis buffer(Thermofisher 89901), 1ul Pierce™ Universal Nuclease(Thermofisher 88700) , 2.5ul Halt™ Protease Inhibitor Cocktail, EDTA-Free(Thermofisher 87785) to the tissue powder, incubated at 4℃ for 1hr.
3. Centrifuge at 13000rpm for 5min, keep the supernatent.

### Western blot detection of GFP and actin protein(internal reference) in plant tissue

1. Add 30ul extracted protein with 10ul 4X SDS-sampling buffer, heat at 95℃ for 5min.
2. Loaded 30ul to the 10% Bio Rad precast PAGE gel. Run at 180V for 50min.
3. Semi-dry transfer at 15V for 30min to a PVDF membrane.
4. 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.
5. Detected by Pierce™ Fast Western Blot Kit, ECL Substrate(Thermofisher, 35050). And record the image with chemi dot instrument.

### Detect the firefly luciferase in plant tissue:

1. Spray the firefly luciferase substract working solution: D-Luciferin, Potassium Salt solution (10 mg/mL in D-PBS) on leaf surface.
2. Incubate at dark for 10min.
3. Image with a chemi dot imager or animal live imager.

### Seed soaking transformation with TTTT:

1. Prepare transformation mixture (1X smart-CD(use the stock one as 20X); 10mM MES buffer PH=5.5; 0.5% Glycerol; 5-10ng/ul endotoxin free transformation vector), incubate at 37℃ for 30min, cool down to room temperature.
2. Clean the seeds with Ethanol/bleach for 30 seconds and wash with distilled water.
3. Add transformation mixture around 1:1 volume ratio with seeds. Incubate at 25℃ for 24hr, change the transformation mixture and incubate another 24hr.
4. Plant the germinated seeds into soil.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

# Results

![](https://static.igem.wiki/teams/5308/icons/team-logo.png)
SZ-SHD

* [Previous page](%7B%7B%20url_for%28%27home%27%29%20%7D%7D)
   [Background](#Background)
   [Challenges on Mars](#Challenges on Mars)
  [Steps toward the solution](#Steps toward the solution)
  [Our design](#Our design)
  [References](#References)

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project and what you learned from them. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort. Furthermore, you can still impress the judges by thoughtfully discussing what went wrong and how you might change things.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}