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