Phaeodactylum tricornutum is a single-celled diatom that lives in the ocean. Diatoms have very efficient photosynthesis and carbon sequestration capabilities, accounting for about 40% of global carbon sequestration and 10-50 times that of forests. It can also utilize both free carbon dioxide and bicarbonate through the CCM mechanism. The aquatic carbon pumping effect based on this mechanism has a great impact on global carbon sinks and can relieve the pressure on atmospheric carbon sinks. Phaeodactylum tricornutum absorb carbon dioxide as well as nitrogen and phosphorus elements in the water column, and use the synergistic effect of carbon, nitrogen and phosphorus in the process of forming a stable carbon sink to form co-precipitation and further enhance the removal of nitrogen and phosphorus elements. We hope to use synthetic biology to construct transgenic algae strains to enhance their carbon sequestration and nitrogen removal and phosphorus removal ability.
We first added waste water to culture microalgae to explore the feasibility of waste water culture of Phaeodactylum tricornutum. At the same time, we overexpressed genes related to carbon sequestration and nitrogen and phosphorus element transport that we screened in the ground organism in an attempt to construct engineered algal strains with greater ability to govern the environment.
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. PHATRDRAFT encodes an ammonium transporter protein that drives the active transport of ammonium into the cell.
We use STUI and KPNI restriction enzymes to digest the pPink-HC-FHG-cp vector, which will be used to introduce into Pichia pastoris. for expression.
In this vector, we added Paox1 promoter, CYC1 terminator, eGFP coding sequence and ampicillin resistance gene. The AOX1 promoter is a common and efficient promoter in Pichia pastoris.. It is a methanol-inducible promoter. It is activated in the methanol carbon source and induces the transcription and translation of the CDS region. When a repressive carbon source such as glucose and glycerol exists, it is activated. The promoter is turned off, inhibiting the transcription and translation of the CDS region. eGFP encodes green fluorescent protein, which can make our transformed Pichia pastoris. cells have green fluorescence.
In the vector used for Phaeodactylum tricornutum transformation, we added ampicillin resistance gene and bleomycin resistance gene. The ampicillin resistance gene can make our transformed Escherichia coli have ampicillin resistance. The bleomycin resistance gene will be that our transformed Phaeodactylum tricornutum has bleomycin resistance, which is convenient for us to screen the transformed Phaeodactylum tricornutum.
Algae strains were cultured separately using seawater and wastewater to verify the ability of the transgenic algae strains to fix carbon and remove nitrogen and phosphorus.