Due to COVID-19, our time in the laboratory has been reduced to some extent. As of October 2022, we have completed most of our experiments. We explored the feasibility of effluent culture of Phaeodactylum tricornutum. And two transgenic algal strains were successfully constructed, and we found that one of the transgenic strains (Pha::CA-PRK-eGFP) had significantly higher lipid content and removal rate of nitrogen and phosphorus elements than the wild type. At the same time, our analysis revealed that the expression levels of genes related to phosphorus transport and lipid metabolism were significantly up-regulated.
CA,CA2,and PRK genes were obtained by nested PCR cloning using cDNA from Phaeodactylum tricornutum as template. After that, these three genes were successfully recombined into the complex plasmid vectors CA-PRK and CA2-PRK to construct the overexpression system. The overexpression system was successfully transferred into the chassis organism for partial functional validation.
We configured the effluent medium in a 1:1 volume ratio of f/2 medium to artificial effluent and cultured wild-type Phaeodactylum tricornutum. The test results were as follows. Cell density: Based on the variation of Phaeodactylum tricornutum density, we found that there was no significant difference between the biomass of Phaeodactylum tricornutum in seawater culture and waste water culture.
Fig1. Cell density
It indicates that the waste water culture of Phaeodactylum tricornutum is feasible.
CA,CA2 and PRK genes were obtained by nested PCR cloning using cDNA of Phaeodactylum tricornutum as template. And CA and CA2 were linked to PRK respectively by linker.
Carbonic anhydrase (CA) plays a key role in CCM and can reversibly catalyze the CO2 hydration reaction. Phosphoribosyl ketokinase (PRK) catalyzes the last step of the Calvin cycle reaction and is important for regulating the flow of sugar in the Calvin cycle.
Fig2. Electrophoresis image of the target gene
The vector we use is pPink-HC-FHG-cp. pPink-HC-FHG-cp is 8678bp in length, contains Paox1 promoter and CYC1 terminator.We use T4 ligase to connect CA-PRK and CA2-PRK to the vector.
Fig3. Overexpression system
The ligated plasmid is transferred to competent cells and cultured. The following is the growth on the medium containing ampicillin.
Fig4. A CA-PRK overexpression system. B CA2-PRK overexpression system.
Pick a single positive colony for culture, and perform PCR detection, and then send the colony with the correct electrophoresis band to the company for sequencing.
Fig5. Colony PCR validation of agarose gel electrophoresis results
According to the sequencing results, the bacterial solution was expanded and cultured and the plasmid was extracted, and the plasmid was verified by PCR detection. Before electroporation transformation, we use the recombinant plasmid as a template for PCR detection to verify whether our recombinant plasmid contains the target gene.
The plasmid was transferred into Pichia pastoris and Phaeodactylum tricornutum using an electroporator.
After screening and culturing, the genomic DNA of Phaeodactylum tricornutum was extracted and PCR was performed to determine whether the recombinant plasmid was successfully transferred into the algal cells. Functional validation of transgenic algae strains.
Fig6. DNA pcr validation agarose gel electrophoresis results
The protein was extracted and the function of the algal strain was verified at the protein level. The results showed that our constructed CA-PRK overexpression system and CA2-PRK overexpression system have been successfully introduced into Phaeodactylum tricornutum.
Fig7. Coomassie bright blue stain
Fig8. Western Blot verify
We also examined the expression of the target genes, and all of them showed a significant increase, which also indicates that we successfully transferred the overexpression system into the algal strain
Fig9. Histogram of gene level expression of target genes
In batch culture, we examined numerous physiological and biochemical parameters such as density, N and P content, lipid content, photosynthetic rate, etc. of wild-type Phaeodactylum tricornutum and Phaeodactylum tricornutum with CA-PRK overexpression system.
It was found that the growth of the transgenic strain was not affected and the biomass was not significantly different from that of the wild-type strain.
Fig10. Cell density line graph
We stained the treatment with Nile Red fluorescent dye and observed it under LSM. The results are as follows. The red area represents chlorophyll and the green area represents lipid. It can be seen that the lipid content of Phaeodactylum tricornutum containing CA-PRK overexpression system is significantly higher than that of wild type.
Fig11. Transgenic microalgae screened for antibiotics
Quantitative analysis revealed that its lipid content was significantly increased, about 2.3 times higher than that of the wild type, and correspondingly its protein content and polysaccharide content were reduced. The increase and decrease in the content of the three metabolites is in line with the widely accepted pattern.
Fig12. Histogram of lipids to dry weight content
Fig13. Glucose content standard curve and Carbonhydrate content histogram
Fig14. Bsa standard curve and protein content histogram
The content of polyunsaturated fatty acids was found to be significantly higher by GC-MS analysis. The content of ARA and EPA were twice as high as that of wild type. DHA content also has some improvement. This makes sense for cost reduction of high value products.
Fig15. Histogram of the content of the three polyunsaturated fatty acids
We found by qpcr that the expression levels of phosphorus transport as well as lipid synthesis-related genes were significantly higher in Phaeodactylum tricornutum containing the CA-PRK overexpression system than in the wild type.
Fig16. qPCR analysis of key genes
Fig17. LCM observation Chart
The growth of the transgenic algae strain did not differ significantly from the wild-type algae strain in the course of the effluent culture.
Fig18. Cell density line graph
Meanwhile, according to the results of Nile red staining, it can be seen that the lipid accumulation of the transgenic algae strain is significantly higher than that of the wild-type algae strain.
Fig19. Nile red fluorescence content line graph
Overall, our transgenic strain was functionally superior to the wild-type strain.
We choose immobilized cell technology. Our experimental materials - Phaeodactylum tricornutum were made into algal spheres using sodium alginate as the embedding material. At the same time, material exchange can still be carried out between the inside and outside of the algal sphere, which will not affect the normal growth of Phaeodactylum tricornutum.
Fig20. Immobilized microalgae actual photo
（The numbers marked in the graph are the number of days of incubation of algal blooms）
It functions well in preventing the escape of algal blooms. No algal plants were observed in the medium at 21 days of incubation.
Fig21. Culture medium observation chart
The use of algal spheres together with our designed photobioreactors can further prevent the escape of algal strains.
During the whole experiment, aseptic operation should be paid special attention. In past experiments, we have had a number of experiments that have failed due to contaminated bacteria. This left us a deep impression.
For the current transgenic algae strain, we are not too comprehensive in testing its physiological and biochemical indicators, and it still has a lot of places worth exploring. In our future work, we will also continue to study its various properties and try our best to make it play more and better roles. Of course, in terms of biosafety, we will also continue to improve the improvement, and strive to design the transfer suicide switch as soon as possible, so that the transgenic algae strains are more safe and controllable.