In the past year, our team took Phaeodactylum tricornutum as the research object in combination with the increasingly tense climate and water ecological environment, hoping to improve the ability of algae to fix carbon, remove nitrogen and phosphorus by means of synthetic biology and alleviate the current environmental problems. Looking back on this year, we have gradually completed the project concept, completed the supporting methods and facilities system, and moved closer to the goal step by step. We hope that our team's history can provide some reference for more teams in the future.
Phaeodactylum tricornutum is a kind of unicellular diatom living in the ocean. It has very effective photosynthesis and carbon storage capacity, accounting for about 40% of the global carbon solid stock, 10-50 times that of forests. At the same time, it can also utilize both free carbon dioxide and bicarbonate through CCM mechanism. The aquatic carbon pump effect based on this mechanism has a great impact on global carbon sink, which can relieve the pressure of atmospheric carbon sink. Phaeodactylum tricornutum can also absorb nitrogen and phosphorus elements in the water body while absorbing carbon dioxide. In the process of forming a stable carbon sink, the synergistic effect of carbon, nitrogen and phosphorus can form coprecipitation to further strengthen the removal of nitrogen and phosphorus elements. We hope to use synthetic biological means to build transgenic algae strains to improve their carbon fixation, nitrogen removal and phosphorus removal capacity.
Fig1. Phaeodactylum tricornutum
In this year's project, we focused on improving the environmental governance capacity of Phaeodactylum tricornutum, especially the ability to fix carbon and remove nitrogen and phosphorus, and constructed three single gene elements. Later, these three genes were successfully recombined into composite plasmid vectors CA-PRK and CA2-PRK to build an over expression system. The overexpression system was successfully transferred into the chassis organism for culture and functional verification. In addition, in order to further understand how the composite plasmid vector works in cells, we optimized the expression of the traditional electroporator of Phaeodactylum tricornutum, and added eGFP tags to it, making it easier to locate genes.
Fig2. Experimental design idea diagram
It can be seen from the experimental results of seawater culture that the growth rate of the transgenic algal strain (Pha:: CA-PRK eGFP) we constructed has not been affected, and the cell density has no significant difference with the wild type algal strain.
Fig3. Cell density line graph
Moreover, the lipid accumulation increased, reaching 2.3 times of that of wild type algae strains. At the same time, the content of polyunsaturated fatty acids in fatty acids increased significantly after GC-MS analysis
Fig4. Histogram of lipids to dry weight content
Fig5. Histogram of the content of the three polyunsaturated fatty acids
Fig6. Transgenic microalgae screened for antibiotics
As for the absorption of nitrogen and phosphorus that we are concerned about, the performance of transgenic algal strain (Pha:: CA-PRK-eGFP) is also as expected, and the absorption rate of nitrogen and phosphorus has been improved
Fig7. Folding graph of the remaining content of nitrogen and phosphorus elements
In the verification of gene expression level, we found that the expression of genes related to nitrogen and phosphorus metabolism and lipid synthesis of Pha::CA-PRK-eGFP was up-regulated, which was significantly different from that of the wild-type alga strain, which supported many results detected by sampling in batch culture.
Fig8. qPCR analysis of key genes
In addition, we successfully located that the target gene we constructed was expressed in the chloroplast of Phaeodactylum tricornutum by using the optimized expression vector.
Fig9. LCM observation Chart
When artificial wastewater is used for culture, the growth of transgenic algal strain (Pha:: CA-PRK-eGFP) is better than that of wild type algal strain, and the lipid accumulation is also significantly increased compared with that of wild type algal strain.
Fig10. Cell density line graph
Fig11. Nile red fluorescence content line graph
Obviously, the construction of our transgenic algae strain is successful. The performance of the transgenic algae strain (Pha::CA-PRK-eGFP) in growth, nitrogen and phosphorus absorption and other aspects is better than that of the wild-type algae strain. It can also be well grown and propagated in wastewater and accumulate high-value components. This makes us a step closer to our proposed goal of using Phaeodactylum tricornutum for water eutrophication treatment and mitigation of greenhouse effect.
We also paid great attention to the biosafety protection of transgenic algae strains. We used the immobilized cell technology and sodium alginate as the embedding material to make algal spheres of Phaeodactylum tricornutum to restrict its growth activities in a certain space. At the same time, the material exchange between the inside and outside of the algal sphere could still be carried out, which would not affect the normal growth of Phaeodactylum tricornutum. The function of the algal spheres in preventing the escape of microalgae was also verified, and no algal strains were observed in the culture medium after 21 days of culture in conical flask.
Fig12. Immobilized microalgae actual photo
（The numbers marked in the graph are thenumber of days of incubation of algal beads）
Fig13. Culture medium observation chart
At the same time, a photobioreactor was designed and manufactured to provide a closed environment for microalgae culture and isolate microalgae from the environment. The bioreactor is equipped with an ultraviolet lamp to inactivate microalgae when necessary. In addition, the equipment is equipped with three water outlets at different positions. In addition to the basic function of changing water, it also has the function of collecting algae beads at different positions.
The above methods and hardware can provide some reference for teams that are difficult to build suicide switches and cannot guarantee biological safety.