iGEM 2020 — Team Worldshaper-Nanjing

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# Team:Worldshaper-Nanjing/Notebook

Worldshaper-Nanjing Notebook

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# Notebook

[Home](https://2020.igem.org/Team%3AWorldshaper-Nanjing)  Experiment [Notebook](https://2020.igem.org/Team%3AWorldshaper-Nanjing/Notebook)

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## Connect Us

WeChat: WorldshaperNJ

Youtube:Worldshaper nanjing

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Facebook：Nanjing Worldshaper

E-mail: worldshapernanjing2020@gmail.com

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# Team:Worldshaper-Nanjing/Engineering

Worldshaper-Nanjing Engineering Success

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# Engineering Success

[Home](https://2020.igem.org/Team%3AWorldshaper-Nanjing)  Experiment [Engineering Success](https://2020.igem.org/Team%3AWorldshaper-Nanjing/EngineeringSuccess)

# Summary

At present, we independently expressed the glucoamylase gene (AnGlu) and α-Amylase gene (AnAmyA) from the Aspergillus niger in the Yarrowia lipolytica Po1g. Their capability for starch degradation was verified by measuring the OD of the two strains in the fermentation medium using the starch as the sole carton source. The results showed that Y. lipolytica expressing AnGlu can degrade the starch efficiently and AnAmyA did not work well. Subsequently, we regulated the C/N ratio by adding different concentrations of starch to verify whether the C/N ratio had the influence on the oil biosynthesis, the results proved that high C/N ratio is more conducive to oil accumulation.

## 1. Plasmid Construction

We constructed the plasmids according to DESIGN page. There are five parts in our plasmid library: a promoter-EXP (BBa\_K3578000), a terminator-XPR2t (BBa\_K3578002), two expression genes-α-amylase (BBa\_K3578003), glucoamylase (BBa\_K3578001), and the Leu selection marker (BBa\_K3578004). By BsaI and SapI IIs enzymes digestion and T4 ligase, we successfully constructed two plasmids which separately carried the AnGlu and AnAmyA expression cassette with Leu selection marker (Figure 1). Then two plasmids were linearizd with EcoR I and transformed into Y. lipolytica plotoplast. The AnGlu/AnAmyA expression cassette with Leu selection marker DNA fragment will be randomly integrated into genome loci.

![Image placeholder](https://static.igem.org/mediawiki/2020/7/79/T--Worldshaper-Nanjing--ES1.png)

Figure 1 Schematic diagram of plasmid construction process.

By the PCR experiment, we randomly selected 10 strains in which AnGlu and Leu expression cassette have been successfully integrated into the genome. In addition, 10 strains integrating AnAmyA and Leu expression cassette were picked up for the following experiments.

![Image placeholder](https://static.igem.org/mediawiki/2020/c/cd/T--Worldshaper-Nanjing--es6.png)

Figure 2 Primer design and agarose gel electrophoresis for plasmid identification

We designed the primers(F/R) to identify the two plasmids. The primer design was shown in the figure 2, and the predicted PCR result is 2736 bp. The electrophoresis results showed that about 3000 bp bands were obtained by using AnGlu/AnAmyA expression cassette as template, the electrophoresis results were consistent with our expectation (Figure 2).

## 2. The expression of AnGlu in Y. lipolytica is sufficient to allow growth on starch

In order to verify whether the AnGlu and AnAmyA possess the ability for starch degradation. Two engineered Y. lipolytica expressing the AnGlu (Polg-AnGlu) and AnAmyA (Polg-AnAmyA) individually were cultured in the starch medium in which the 30 g/L starch was used as the sole carton source. In addition, the strain Po1g was also cultured in the starch medium with the addition of 0.4g/L leucine as the control (Polg-Leu). The OD was measured every 24 hours. As shown in Figure 3, only Po1g-AnGlu strain can grow well in the starch medium, it means that AnGlu had the relatively higher starch utilization efficiency. On the contrary, same as the control, the Polg-AnAmyA almost did not growth in the starch medium. The result can also be directly observed as shown in Figure 4, in which Polg-AnGlu possess a much higher cell density. In summary, Po1g-AnAmyA did not work as expected, but the expression of AnGlu in Y. lipolytica is sufficient to allow growth on starch.

![Image placeholder](https://static.igem.org/mediawiki/2020/8/8a/T--Worldshaper-Nanjing--ES2.png)

Figure 3 Growth curve in starch medium as sole carbon source

![Image placeholder](https://static.igem.org/mediawiki/2020/2/27/T--Worldshaper-Nanjing--ES3.png)

Figure 4 Photos of Y. lipolytica solution after culturing 96 hours.

## 3. High C/N ratio is more conducive to oil accumulation

Since the Polg-AnGlu strain can growth well on the starch medium, we then measured the lipid titer and content. The Polg-AnGlu can produce up to 0.76 ± 0.04 g/L lipid and 14.66 ± 0.43% of DCW as fatty acids in the starch media and a C/N ratio of 30. The titer and content were relatively low. Previous researchers proved that higher C/N ratio is more conducive to lipid accumulation when glucose, xylose and glycerin were used as the carbon source. In order to verify whether the higher C/N ratio in the starch media also help improve the lipid biosynthesis capability, we regulated the C/N ratio by adding different concentrations of starch (Figure 5). The C/N ratio was 30, 60, 90. These statistics showed that when C/N ratio was increased to 60, the lipid titer increased from 0.76 ± 0.04 g/L to 1.07 ± 0.02 g/L, a 55% improvement. when C/N ratio was increased to 90, the lipid titer further increased to 1.17 ± 0.02 g/L. Similar results about the increasing lipid content were also observed. The lipid content increased from 14.66 ± 0.43% to 24.06 ± 0.13% of DCW, a 64% improvement. In summary, it shows that improving C/N ratio is an effective strategy to strengthen lipid accumulation.

![Image placeholder](https://static.igem.org/mediawiki/2020/2/2b/T--Worldshaper-Nanjing--ES4.png)

Figure 5 Lipid production and content in the starch medium with different C/N ratio

## 4. Future work

Due to the Covid-19 epidemic, the experimental time of this project is limited, and lots of following plans have not yet been carried out. According to the experimental results, we are ready to further increase AnGlu gene copy number to accelerate starch degradation rate. In addition, the strategies for improving the lipid titer, yield and rate were also taken into consideration, including disrupting the fatty acid oxidation pathway, increasing the content of acetyl coenzyme A in cytoplasm, simultaneously overexpressing the rate-limiting genes-diacylglycerol acyltransferase and acetyl-CoA carboxylase, as well as balancing the cofactor supply.

At last, according to the Human Practices survey results, we found that the biggest contradiction in the production of biodiesel and even biofuels is the lack of cheap raw materials. Therefore, in the future, we need to further expand the scope of our usable waste substrates to better realize the sustainable development goals of protecting the global environment and waste utilization.

## Connect Us

WeChat: WorldshaperNJ

Youtube:Worldshaper nanjing

Bilibili：WorldshaperNJ

Weibo：WorldshaperNJ

Facebook：Nanjing Worldshaper

E-mail: worldshapernanjing2020@gmail.com

## Links

* [Home](https://2020.igem.org/Team%3AWorldshaper-Nanjing)
* Project
* Experiment
* Dry Lab
* [Part](https://2020.igem.org/Team%3AWorldshaper-Nanjing/Parts)
* Team

## QR code

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# Team:Worldshaper-Nanjing/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:Worldshaper-Nanjing/Results

# Results

You can describe the results of your project and your future plans here.

### 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](http://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. 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.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)