In our project, we focused on enhancing the environmental management ability of Phaeodactylum tricornutum by constructing five new parts and successfully recombined these five parts into plasmid vectors to construct an overexpression system. The overexpression system was then transferred to Phaeodactylum tricornutum for functional validation. Finally, the transformation results were tested and validated to investigate whether the overexpression system was successfully expressed in algae strains, the effect on its growth and metabolism, and whether its carbon sequestration capacity was enhanced.
Fig1. Flow Chart
At the beginning of the project, we wanted to use Phaeodactylum tricornutum to treat waste water. Associating its strong carbon sequestration ability, we wanted to construct engineered algal strains with stronger environmental management ability to help mitigate climate problems. After reviewing the documents, we targeted the following genes to try to construct a recombinant plasmid system.
1. Gene acquisition: Using the cDNA of P. tricornutum as a template, the PTCA-encoded carbonic anhydrase of P. tricornutum was cloned by overlap PCR. As a member of the β-CA family, it can reversibly catalyze the CO2 hydration reaction, which can improve CO2 conversion efficiency and play a key role in CCM, as well as in ion exchange, CO2 acquisition, and photosynthesis.
PTCA2, which is highly homologous to PTCA, encodes a protein with fully conserved β-CA zinc coordination residues and significant levels of mRNA accumulation at low CO2 concentrations.
PRK encodes phospho-ribulose kinase, an enzyme specific to the Calvin cycle, which plays a crucial role in regulating the flow of sugars in the Calvin cycle by catalyzing the formation of the receptor ribulose-1,5-bisphosphate of CO2 with the help of ATP phosphorylation.
Fig2. Electrophoresis image of the target gene
2. Ligation: The vector we used is pPink-HC-FHG-cp. pPink-HC-FHG-cp is 8678bp in length and contains the Paox1 promoter and CYC1 terminator. We used T4 ligase to ligate CA, CA2, PRK ,CA-PRK and CA2-PRK to the vector, respectively.
Fig3. Overexpression system
At the beginning, we did not insert eGFP tags in the overexpression system, but after talking with experts and reviewing documents, we decided to insert eGFP for labeling to have a better observation and verification of the transfer and expression of our recombinant plasmids.
Fig4. A CA-PRK overexpression system. B CA2-PRK overexpression system.
1.Transformation: The constructed plasmids are transferred into receptor cells for culture.
2.Validation: Pick off single colonies for incubation and PCR, select the correct bands for agarose gel electrophoresis and sequencing.
Fig5. Colony PCR validation
3. Plasmid acquisition: Based on the sequencing results, the culture is expanded, the plasmid is extracted, and the plasmid is verified to contain the target gene by PCR assay.
1.Electroporation: use electroporation protocol to transfer the plasmid into Phaeodactylum tricornutum.
Fig6. Transgenic algae strain screening
2.Validation: After screening and culturing, extract the genomic DNA of Phaeodactylum tricornutum and determine whether the recombinant plasmid is successfully transferred into the algal cells by PCR.
Fig7. DNA level validation
Extract proteins to verify algal plant function from the protein level.
Fig8. Coomassie bright blue stain
Fig9. Western Blot verify
Target gene expression validation
Fig10. Gene level expression of target genes
We used seawater f/2 medium to cultivate transgenic algae strains, and tested many physiological and biochemical indicators. The test results are as follows.
In terms of growth, the growth of the algae strains transferred into the overexpression vector system was not affected, and there was no significant difference between their cell density and that of wild type algae strains.
Fig11.Cell density
Chlorophyll fluorescence parameters
Fig12.Photosynthetic rate on day 5 of incubation
Lipid content and Lipid composition
Fig13. Transgenic microalgae screened for antibiotics
After testing, both transgenic algae strains were found to have significantly higher oil content. Pha::CA-PRK-eGFP has 2.3 times the oil content of the wild type.
Fig14. Fatty acid composition analysis
Fig15. Nitrogen and phosphorus content
The results showed that the expression levels of genes related to phosphorus transport and lipid metabolism were significantly up-regulated in one of the transgenic algae strains (Pha:: CA-PRK-eGFP).
Fig16. qPCR analysis of key genes
Based on the above indexes, we screened out a better algal strain (Pha:: CA-PRK-EGFP) for waste water culture to verify its viability in wastewater and nitrogen and phosphorus removal capacity.
After testing, we found that the growth of transgenic algae was not affected under the waste water culture environment, and the accumulation of lipid was significantly increased.
Fig17. Cell density
Fig18. Nile red fluorescence content