iGEM 2025 — Team aei-prep-taiwan

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# DBTL Cycle

##

![DBTL Cycle Diagram](https://static.igem.wiki/teams/5744/engineering/dbtl-cycle.webp)

Figure I. DBTL cycle

Our DBTL cycle was designed as a plasmid to mimic our project where we used plasmids to genetically engineer E. huxleyi and increase bio-fuel and bio-concrete production. We employed the DBTL cycle in all parts of our experimental process. This cycle helped us improve, organize and optimize our experimental strategies to enhance the effects of our plasmids on three chosen genes.

## Design

For our experimental design, we engineered three genes, CA9, NCE103, and DGA1, and under control of the CMVp promoter we cloned them into the backbone of plasmid Y312. These plasmids were then inserted into E. huxleyi to transform our algae into a biofactory for the production of bio-fuel and bio-concrete. Simultaneously we analyzed production analysis and engaged in mass production of our transformed algae.

Post-transformation, E. huxleyi did not grow to our expectations in order to properly evaluate bio-concrete and bio-fuel production, therefore, we switched to a yeast model to determine if the modified genes were working as intended.

![DBTL Cycle Diagram](https://static.igem.wiki/teams/5744/plant/tag-pathway-yeast.avif)

Figure II: the TAG synthesis pathway in yeast (Sharma et.al., 2018)

## Build

Using our experimental design above, we inserted our three genes of interest into the CMV promoter. We then cloned them into the backbone of our plasmid Y312. To determine if our genes of interest were in the plasmid, we performed restriction enzyme digestions and gel electrophoresis. We determine that our genes of interest were successfully integrated into the plasmid. We then transformed our algae with the Y312 plasmid and verified successful integration with gel electrophoresis.

To show proof of concept, we performed lipid experiments on yeast, a biological system known for its rapid reproduction cycle and the capability to produce lipids. We used DH5a E. coli to insert our genes of interest into yeast for further testing and analysis. Post-transfection we performed restriction enzyme digestion and gel electrophoresis to verify our genes of interest were successfully integrated into the yeast genome.

## Test

Tests in our algae were inconclusive as the algae did not grow to the standards we had set out for the experiment. Therefore, we couldn’t test the ability of algae to respond to temperature, light or sound to increase production of bio-fuel and bio-concrete. Tests for coccolith growth were inconclusive.

In order to show proof of concept that our genes of interest produce lipids capable of being extracted for bio-fuel, we performed several lipid extraction experiments to look for the effect of the genes on total lipid production, which were subsequently verified and quantified via weighing.

## Learn

Our algae experiments showed successful integration of our genes of interest; however, algae production, bio-fuel, and bio-concrete production were unsuccessful. We believe that we need to order additional algae and perform the experiments again. We know that the plasmids were successfully created and inserted into algae; therefore, we believe that environmental conditions may have affected this part of our experiment and prevented the production of algae, bio-fuel, and bio-concrete.

When we inserted our genes of interest in yeast, we found that gene DGA1 successfully increased oil production in one of our oil extraction experiments; however, we believe that a significant loss of hexane during the experiment may have contributed to some loss of oil in our experiments. Therefore, it is reasonable to believe that if hexane had not been lost during the oil extraction experiments, oil production may have markedly increased across all oil extraction experiments. In the future, we will give this consideration when extracting oil from yeast.

# Gene Design

##

![CMVp Genes](https://static.igem.wiki/teams/5744/engineering/gene-design.webp)

Figure III. CMVp Driven Genes

Our gene design was based on known biochemical pathways in E. huxleyi. There are several currently known biochemicals that could be exploited in our algae to create bio-fuel and bio-concrete. We chose to select the triacylglycerol pathway to target and increase oil production, and we used the coccolith formation pathway to target calcite production. According to multiple literature sources, the detailed pathway for calcite production has not been fully elucidated at this time; however, based on the biochemical properties of calcite, we hypothesized that carbonate anhydrase plays a crucial role in coccolith formation. This is why we chose genes NCE103 and CA9 as overexpression targets for increasing calcite production. We enlisted TWIST bioscience to assist with gene fragment generation and synthesis on commercial plasmid pTWIST. We further cloned pTWIST into our overexpression vector Y312.

# Plasmid Design

##

![y312 plasmid](https://static.igem.wiki/teams/5744/engineering/plasmid-backbone.webp)

Figure IV. Y312 Plasmid Backbone

We used two expression systems in our vector design. The first system utilized CMVp driven overexpression of our genes of interest, and the second system utilized pLac2 driven overexpression in yeast for proof of concept.
We designed the CMV promoter as follows: CMVp-MCS-IRES-NeoR. We chose this order to prevent potential methylation silencing of the CMV promoter and selecting the target clone easier. We used DH5a E. coli to clone our synthesized gene fragments into our expression vectors and express the target fragments.

# References

DGA1. DGA1 | SGD. (n.d.). https://www.yeastgenome.org/locus/S000005771

Florentin, D., Fontana, A., Baccou, J. C., & Strub, C. (2014, September). (PDF) Single Cell Oils (scos) from oleaginous yeasts and moulds: Production and Genetics. Single cell oils (SCOs) from oleaginous yeasts and moulds: Production and genetics. https://www.researchgate.net/publication/263856626\_Single\_cell\_oils\_SCOs\_from\_oleaginous\_yeasts\_and\_moulds\_Production\_and\_genetics

NCE103. NCE103 | SGD. (n.d.). https://www.yeastgenome.org/locus/S000004981

U.S. National Library of Medicine. (2025, August 19). Ca9 carbonic anhydrase 9 [homo sapiens (human)] - gene - NCBI. National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/gene/768

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# Experimental Overview

![Experimental Overview Diagram](https://gitlab.igem.org/2025/aei-prep-taiwan/-/raw/4409b27123e1fb9ca4d9dbce3c6d709b40137c3d/static/Experiment_Overview.png)

##

The main focus of our project is to genetically modify Emiliana huxleyi to increase carbon fixation efficiency by increasing the amount of lipids and calcite they produce in a period of time. To directly improve the efficiency of lipid production, our team engineered DGA1. To improve the efficiency of calcite production, CA9 and NCE103 were chosen. Along with the control of CMV promoter, finally cloning into the Y312 plasmid backbone. In theory, with the expression of CA9, NCE103, and DGA1 genes, the E. huxleyi carbon fixation rate into lipids and coccoliths will be significantly increased under specific environmental conditions.

# Experiments

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[Algae Tank Culture](https://static.igem.wiki/teams/5744/experimentspdf/algae-tank-culture.pdf)

[Algae Vial Culture](https://static.igem.wiki/teams/5744/experimentspdf/algae-vial-culture.pdf)

[E Huxleyi Culture Medium](https://static.igem.wiki/teams/5744/experimentspdf/e-huxleyi-culture-medium.pdf)

[Gel Electrophoresis](https://static.igem.wiki/teams/5744/experimentspdf/gel-electrophoresis.pdf)

[Gel Extraction From Electrophoresis](https://static.igem.wiki/teams/5744/experimentspdf/gel-extraction-from-electrophoresis.pdf)

[Ligation Transformation](https://static.igem.wiki/teams/5744/experimentspdf/ligation-transformation.pdf)

[Lipid Extraction Trial 1](https://static.igem.wiki/teams/5744/experimentspdf/lipid-extraction-trial-1.pdf)

[Lipid Extraction Trial 2](https://static.igem.wiki/teams/5744/experimentspdf/lipid-extraction-trial-2.pdf)

[Mass Lipid Extraction](https://static.igem.wiki/teams/5744/experimentspdf/mass-lipid-extraction.pdf)

[PCR Amplification](https://static.igem.wiki/teams/5744/experimentspdf/pcr-amplification.pdf)

[Plasmid Design](https://static.igem.wiki/teams/5744/experimentspdf/plasmid-design.pdf)

[Research Audio Choices](https://static.igem.wiki/teams/5744/experimentspdf/research-audio-choices.pdf)

[Restriction Enzyme Digestion](https://static.igem.wiki/teams/5744/experimentspdf/restriction-enzyme-digestion.pdf)

[Subclone Synthetic Genes Into Expression Plasmid](https://static.igem.wiki/teams/5744/experimentspdf/subclone-synthetic-genes-into-expression-plasmid.pdf)

# References

##

Radakovits, R., Jinkerson, R. E., Darzins, A., & Posewitz, M. C. (2010). Genetic Engineering of Algae for Enhanced Biofuel Production. Eukaryotic Cell, 9(4), 486–501. https://doi.org/10.1128/ec.00364-09

DGA1. DGA1 | SGD. (n.d.). https://www.yeastgenome.org/locus/S000005771

NCE103. NCE103 | SGD. (n.d.). https://www.yeastgenome.org/locus/S000004981

U.S. National Library of Medicine. (2025, August 19). Ca9 carbonic anhydrase 9 [homo sapiens (human)] - gene - NCBI. National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/gene/768

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## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/results)
* [2024 Marburg](https://2024.igem.wiki/marburg/results)
* [2024 EPFL](https://2024.igem.wiki/epfl/results)
* [2024 UToronto](https://2024.igem.wiki/utoronto/results)
* [2024 Marburg](https://2024.igem.wiki/marburg/results)
* [2024 Sydney-Australia](https://2024.igem.wiki/sydney-australia/results)
* [2024 Toulouse-INSA-UPS](https://2024.igem.wiki/toulouse-insa-ups/results)
* [2024 BNDS-China](https://2024.igem.wiki/bnds-china/results)
* [2024 Vilnius-Lithuania](https://2024.igem.wiki/vilnius-lithuania/results)
* [2024 XMU-China](https://2024.igem.wiki/xmu-china/results)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/results)

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