A genetically engineered algae with controllable flocculation, and its construction method and application

Abstract

The invention discloses a genetically engineered algae with controllable flocculation, a construction method and an application thereof. The invention is characterized in that the genetically engineered algae is a triangular shaped algae into which an enabling plasmid pPha-J47869-EG01992-eGFP-FSP is introduced, and the FSP is directionally expressed on the outside of the triangular shaped algae cells. The construction method comprises the steps of constructing an inducible expression plasmid pPha-J47869-eGFP and a directional expression plasmid pPha-J47869-EG01992-eGFP; artificially synthesizing a codon-optimized FSP coding sequence and inserting it into the downstream of the eGFP of the directional expression vector to construct the enabling plasmid; and finally, electro-transforming the enabling plasmid into the triangular shaped algae, coating the algae on an f / 2 plate culture medium for culture, and screening and identifying positive algae strains to construct the genetically engineered algae with controllable flocculation. The invention has the advantages of being able to efficiently combine with white carbon black, and efficiently coupling the enabling process of the microalgae with its culture and collection process.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant breeding, and in particular relates to a controllable flocculation genetically engineered algae and a construction method and application thereof. Background Art

[0002] Microalgae have a far higher photosynthetic carbon fixation efficiency than higher plants and can produce large quantities of high-value-added substances such as polyunsaturated fatty acids, astaxanthin, and fucoxanthin, offering enormous economic development potential. Microalgae, represented by diatoms, contain fucoxanthin content tens or even hundreds of times higher than that of large brown algae, and the large-scale production of fucoxanthin has attracted widespread attention. However, the mass density of microalgae cells is similar to that of water, and their tiny size makes them difficult to separate from the water column after high-density cultivation. Therefore, the efficient collection of microalgae cells from culture water has become a bottleneck restricting the development of the microalgae industry.

[0003] Although there are currently a variety of cell collection methods, such as flocculation, microfiltration, plate and frame filtration, flotation, and high-speed centrifugation, none of these existing methods can achieve efficient collection of microalgae cells while saving energy. Take high-speed centrifugation, the most mainstream cell collection method in the microalgae industry, as an example: centrifugal collection relies on expensive large centrifuges, which not only require huge initial investment but also high maintenance costs. In addition, centrifugal energy consumption is extremely high, and operating costs are very high. It is estimated that the cost of centrifugal collection even reaches more than one-third of the total cost of microalgae production. Therefore, the development of a new collection system that is efficient, safe, and low-energy is a key issue that the microalgae industry needs to solve urgently.

[0004] Cell-enabling technology involves introducing specific biomolecules with specialized functions into target cells through genetic engineering and other means for expression, thereby endowing the target cells with specific new functions. Furthermore, the introduction of an inducible expression system allows for the efficient coupling of microalgae enabling processes with their cultivation and collection, significantly improving their overall performance in biotechnology applications. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a controllable flocculation genetically engineered algae capable of efficiently coupling the enabling process of microalgae with its cultivation and collection process, as well as a construction method and application thereof.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a genetically engineered algae with controllable flocculation, wherein the genetically engineered algae is a triangular shaped algae into which the enabling plasmid pPha-J47869-EG01992-eGFP-FSP is introduced, and the FSP ( Functional Silica-binding peptide, functional silicon-binding peptide) has the ability to efficiently and specifically bind to silica, and can be directed to be expressed on the outside of the cells of Phaeodactylum tricornutum through fusion expression with EG01992, while the J47869 promoter can exhibit high transcriptional activity under low phosphate concentration conditions. The nucleotide sequence of the enabling plasmid pPha-J47869-EG01992-eGFP-FSP is shown in SEQ ID NO.1.

[0007] The present invention also provides a method for constructing the above-mentioned genetically engineered algae capable of controllable flocculation, comprising the following steps:

[0008] Step 1: Using the genomic DNA of Phaeodactylum tricornutum as a template, the Phatr3_J47869 promoter was amplified and replaced with the FcpA promoter of the basic expression plasmid pPha-T1-eGFP to construct the inducible expression plasmid pPha-J47869-eGFP;

[0009] Step 2: Using the genomic DNA of Phaeodactylum tricornutum as a template, the coding sequence of Phatr3_EG01992 was amplified and inserted upstream of the eGFP of the inducible expression vector to construct the directional expression plasmid pPha-J47869-EG01992-eGFP;

[0010] Step 3: artificially synthesize the codon-optimized FSP coding sequence and insert it into the downstream of eGFP in the directional expression vector to construct the enabling plasmid pPha-J47869-EG01992-eGFP-FSP;

[0011] Step 4: electro-transform the enabling plasmid pPha-J47869-EG01992-eGFP-FSP into Phaeodactylum tricornutum, and spread the transformed Phaeodactylum tricornutum cells on f / 2 plate culture medium for culture, and screen positive algae strains to obtain genetically engineered algae with controllable flocculation.

[0012] Furthermore, step 1 is as follows:

[0013] (1) Based on the Phatr3_J47869 promoter sequence, a forward amplification primer containing an NdeI restriction site and a reverse amplification primer containing an EcoRI restriction site were designed. PCR amplification was performed using P. tricornutum DNA as a template to obtain a Phatr3_J47869 promoter amplification product. The nucleotide sequence of the Phatr3_J47869 promoter forward amplification primer is shown in SEQ ID NO. 3: 5'-GTACTGAGAGTGCACCATATGTGGTGGTGAATCAACACTTAATGTG-3', and the nucleotide sequence of the Phatr3_J47869 promoter reverse amplification primer is shown in SEQ ID NO. 4: 5'-TTAGTCGATGATATCGAATTCCGAAGAATTCTTTTCACCAGGG-3';

[0014] (2) The original FcpA promoter of pPha-T1-eGFP was removed using NdeI and EcoRI restriction endonucleases; the amplified product of the Phatr3_J47869 promoter was connected with the enzyme-cleaved product of pPha-T1-eGFP to construct the inducible expression plasmid pPha-J47869-eGFP.

[0015] Furthermore, step 2 is as follows:

[0016] (1) Forward and reverse primers containing KpnI restriction sites were designed based on the coding sequence of Phatr3_EG01992. PCR amplification was performed using the DNA of Phaeodactylum tricornutum as a template to obtain the amplified product of the coding sequence of Phatr3_EG01992.

[0017] The nucleotide sequence of the primer for forward amplification of the Phatr3_EG01992 coding sequence is shown in SEQ ID NO. 6: 5′-GACTAATTCGAGCTCGGTACCATGAGGCTACGTTCATCCATTACC-3′, and the nucleotide sequence of the primer for reverse amplification of the Phatr3_EG01992 coding sequence is shown in SEQ ID NO. 7: 5′-TCTAGAGGATCCCCGGGTACCACGGCAAAAGATTCCAAAACG-3′;

[0018] (2) The pPha-J47869-eGFP plasmid was digested with KpnI restriction endonuclease, and the amplified product of the Phatr3_EG01992 coding sequence was ligated with the digested pPha-J47869-eGFP to construct the directional expression plasmid pPha-J47869-EG01992-eGFP.

[0019] Furthermore, step 3 is as follows:

[0020] (1) Synthesis of double-stranded template: The coding sequence of the FSP short peptide was codon-optimized to obtain the nucleotide sequence of the FSP coding gene as shown in SEQ ID NO.9: 5'-ATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3'. FSP double-stranded forward and reverse synthesis primers were designed based on the FSP coding sequence and the flexible linker coding sequence. The two synthesis primers were synthesized into double-stranded nucleotides as PCR amplification templates by annealing reaction. The nucleotide sequence of the FSP double-stranded forward synthesis primer was shown in SEQ ID NO.10: 5'-GGCGGCGGTGGTAGCGGTGGCGGCGGTTCCATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3', and the nucleotide sequence of the FSP double-stranded reverse synthesis primer was shown in SEQ ID NO.11: 5'-TTACCGCACAGCAGCCGACATGAGGTCGCTGCGGTGCATGGAACCGCCGCCACCGCTACCACCGCCGCC-3'.

[0021] (2) Design FSP forward and reverse amplification primers based on the XbaI restriction site sequence and the FSP coding sequence, and use the double-stranded nucleotide obtained in step (1) as a template for PCR amplification to obtain an amplification product containing the FSP coding sequence, the corresponding restriction site and the linker coding sequence, wherein the nucleotide sequence of the FSP forward amplification primer is shown in SEQ ID NO.12: 5'-GACGAGCTGTACAAGTCTAGGGCGGCGGTGGTAGCGGT-3', and the nucleotide sequence of the FSP reverse amplification primer is shown in SEQ ID NO.13: 5'-TGCCTGCAGGTCGACTCTAGATTACCGCACAGCAGCCGA-3';

[0022] (3) The pPha-J47869-EG01992-eGFP plasmid was digested with XbaI restriction endonuclease, and the FSP amplification product was ligated with the digested pPha-J47869-EG01992-eGFP to construct the enabling plasmid pPha-J47869-EG01992-eGFP-FSP, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0023] Furthermore, step 4 is as follows:

[0024] (1) Transformation of Phaeodactylum tricornutum: The enabling plasmid pPha-J47869-EG01992-eGFP-FSP was transformed into competent cells, and after incubation, the cells were spread onto LB solid culture medium; after visible colonies were formed, the colonies were picked and transferred to LB liquid culture medium for shaking culture; the transformed competent cells were collected, the enabling plasmid pPha-J47869-EG01992-eGFP-FSP was extracted, and the cells were transformed into Phaeodactylum tricornutum by electroporation;

[0025] (2) Screening of positive transformants: The transformed P. tricornutum cells were spread on a solid f / 2 plate culture medium containing bleomycin and placed under a light intensity of 50-70 µmol photons m -2 s -1 , culture under the conditions of temperature of 20-24℃, screen the positive transformation columns, and obtain the genetically engineered algae with controllable flocculation.

[0026] The present invention also provides the use of the above-mentioned controllable flocculation genetically engineered algae in preparing a microalgae flocculation system. The microalgae flocculation system is an algae liquid added with white carbon black, and the amount of white carbon black added is 0.5-1.5g per liter of P. tricornutum culture liquid.

[0027] Compared to existing technologies, the present invention offers advantages in that: The present invention provides a genetically engineered algae capable of controllable flocculation, as well as its construction method and application. The amino acid sequence MHRSDLMSAAVR (FSP) exhibits a high affinity for silica. Expressing this fragment on the extracellular side of microalgae can confer the ability to efficiently bind silica. The Phatr3_EG01992 gene encodes a protein localized to the extracellular side of microalgae. Fusion expression of this protein with FSP enables targeted expression of FSP on the extracellular side of microalgae. During microalgae cultivation, the nutrient content in the culture system decreases rapidly as cell density increases. By the time the cell density reaches a suitable level for harvesting, the nutrient content in the culture system is typically depleted. Placing Phatr3_EG01992 and FSP under the control of a promoter induced by low nutrient concentrations allows synchronization of FSP's targeted expression on the extracellular side of microalgae with optimal harvesting time. Placing the Phatr3_EG01992-FSP fusion gene under the control of the Phatr3_J47869 promoter allows for efficient coupling of the FSP-enabling process with the cultivation and collection of microalgae. Adding silica to the culture system, due to its much greater density than water, allows the microalgae cells bound to silica to sink rapidly to the bottom of the water, enabling efficient collection of the microalgae.

[0028] In summary, the present invention provides a genetically engineered algae with controllable flocculation, a construction method, and an application thereof. By utilizing the ability of FSP to specifically and efficiently bind to silica, the property of Phatr3_EG01992 localized outside the cell, and the characteristic that the promoter activity of Phatr3_J47869 is regulated by phosphate concentration, the microalgae species are genetically improved. The microalgae germplasm transformed by this strategy can use silica as a flocculant to construct an efficient, low-energy flocculation system, thereby providing new ideas for overcoming the pain points in the microalgae industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 To show the changes in fluorescence intensity of green fluorescent protein (eGFP) under different phosphate concentration conditions after the green fluorescent protein (eGFP) coding sequence was placed under the control of the Phatr3_J47869 promoter;

[0030] Figure 2 This is the electrophoresis diagram of the amplified Phatr3_J47869 promoter, where lane 1: 5000 bp marker, lanes 2-3: Phatr3_J47869 promoter amplification results;

[0031] Figure 3 This is the electrophoresis diagram of the amplified Phatr3_EG01992 coding sequence, where lane 1: 2000 bp marker, lanes 2-3: amplification results of the Phatr3_EG01992 coding sequence;

[0032] Figure 4 The results of screening positive transformants of Phaeodactylum tricornutum using the plate method with the help of antibiotics;

[0033] Figure 5 The sequence results are obtained after DNA from the transformed algae strain was amplified using specific primers based on the eGFP coding sequence.

[0034] Figure 6 To compare the transcription of Phatr3_EG01992 in wild-type strains, strains transformed with the inducible plasmid pPha-J47869-eGFP, and strains transformed with the enabling plasmid pPha-J47869-EG01992-eGFP-FSP based on real-time quantitative PCR.

[0035] Figure 7 To compare and analyze the eGFP signals in wild-type strain, induction plasmid pPha-J47869-eGFP transformant, and enabling plasmid pPha-J47869-EG01992-eGFP-FSP transformant based on laser confocal microscopy.

[0036] Figure 8This is a comparative analysis of the binding of Phaeodactylum tricornutum cells and wild strains with silica based on the enabling plasmid pPha-J47869-EG01992-eGFP-FSP. DETAILED DESCRIPTION

[0037] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0038] Microalgae: Phaeodactylum tricornutum; basic plasmid: pPha-T1-eGFP; amplification kit: 2×Phanta FlashMaster Mix (Dye Plus) (Vazyme, P520-01); competent cells: DH5α; ligation kit: ClonExpress® II One Step Cloning Kit (Vazyme, C112-02); DNA extraction: 2×CTAB (Coolaber, SL2071); plasmid extraction kit: FastPure Plasmid Mini Kit (Vazyme, DC201-01), etc.; the culture medium used was according to the literature (Guillard RRL, Ryther JH. Studies of marine planktonic diatoms:I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran[J]. Canadian Journal of Microbiology, 1962, 8(2): 229–239) with f / 2 culture medium; the microalgae culture equipment was a Zhichu ZQZY-88CGES full-temperature shaking incubator; the microalgae culture conditions were: light intensity 60 µmol photons m -2 s -1 , temperature 22°C, and shaker speed 90 rpm.

[0039] Different genetic transformation systems, different promoters, different positioning tags, different functional peptides and corresponding affinity materials for each microalgae can all be used for the microalgae germplasm transformation based on the cell enabling strategy mentioned in this patent and the subsequent flocculation system construction. This specific embodiment only lists the use of triangular flavonoids as research materials, the use of electroporation method, FSP as the enabling factor, the use of Phatr3_J47869 promoter to achieve induction enabling, and Phatr3_EG01992 as the positioning tag to construct a genetically engineered triangular flavonoids introduced with the enabling plasmid pPha-J47869-EG01992-eGFP-FSP, and white carbon black as the flocculation medium to establish a corresponding flocculation collection system, wherein the nucleotide sequence of the enabling plasmid pPha-J47869-EG01992-eGFP-FSP is shown in SEQ ID NO.1.

[0040] Specific Example 1: Construction of inducible expression plasmid pPha-J47869-eGFP.

[0041] The promoter activity of the Phatr3_J47869 gene is regulated by phosphate concentration and exhibits high transcriptional activity under low phosphate conditions, e.g. Figure 1 As shown. Therefore, by controlling the expression of Phatr3_EG01992 and the enabling factor FSP through the Phatr3_J47869 promoter, the directional expression of FSP on the outside of the microalgae cells is triggered when the phosphate concentration in the culture system decreases. The construction method of the inducible expression plasmid pPha-J47869-eGFP includes the following steps:

[0042] Step 1: Extract genomic DNA of Phaeodactylum tricornutum: Collect cells in the logarithmic phase (cell density 5×10 6 cells / mL) of Phaeochromis tricornutum algae solution, and genomic DNA was extracted;

[0043] Step 2, PCR amplification: According to the Ensembl Protists database, the upstream sequence of the open reading frame of Phatr3_J47869 was searched, and its promoter sequence was predicted with the help of Promoter2.0. The nucleotide sequence of the Phatr3_J47869 promoter was obtained as shown in SEQ ID NO.2. Based on the Phatr3_J47869 promoter sequence, a forward amplification primer containing an NdeI restriction site and a reverse amplification primer containing an EcoRI restriction site were designed. The nucleotide sequence of the forward amplification primer of the Phatr3_J47869 promoter is shown in SEQ ID NO.3: 5'-GTACTGAGAGTGCACCATATGTGGTGGTGAATCAACACTTAATGTG-3', and the nucleotide sequence of the reverse amplification primer of the Phatr3_J47869 promoter is shown in SEQ ID NO.4: 5'-TTAGTCGATGATATCGAATTCCGAAGAATTCTTTTCACCAGGG-3';

[0044] The PCR amplification system was as follows: 0.5 μL of Phaeodactylum tricornutum DNA, 10 μL of 2×Phanta Flash Master Mix, 0.5 μL each of the forward and reverse amplification primers of the Phatr3_J47869 promoter, and 8.5 μL of ddH2O;

[0045] The PCR amplification program was as follows: 94°C for 5 min; 94°C for 30 sec, 68°C for 30 sec, and 72°C for 1 min, 35 cycles, and storage at 4°C;

[0046] Step 3, PCR amplification product verification: After amplification, the PCR products were separated and verified by agarose gel electrophoresis. Figure 2 As shown, lane 1 is a DNA marker; lanes 2 and 3 are amplified Phatr3_J47869 promoter sequences, which were subsequently recovered by gel excision and sequenced. The length of the amplified fragments is consistent with the theoretical length.

[0047] Step 4: Use NdeI and EcoRI restriction enzymes to remove the original FcpA promoter of pPha-T1-eGFP; ligate the Phatr3_J47869 promoter amplification product obtained in step 3 with the enzyme-cleaved product of pPha-T1-eGFP, react at 37°C for 30 min, and construct the inducible expression plasmid pPha-J47869-eGFP.

[0048] Specific Example 2: Construction of directional expression plasmid pPha-J47869-EG01992-eGFP.

[0049] The protein encoded by Phatr3_EG01992 is localized to the outside of microalgae cells. Fusion expression of this protein with FSP can achieve targeted expression of FSP outside of microalgae cells. The construction method of the targeted expression plasmid pPha-J47869-EG01992-eGFP includes the following steps:

[0050] Step 1. Extraction of genomic DNA from Phaeodactylum tricornutum: Collect cells in the logarithmic phase (cell density 5×10 6 cells / mL) of Phaeodactylum tricornutum algae solution, and genomic DNA was extracted;

[0051] Step 2, PCR amplification: According to the Ensembl Protists database, the open reading frame sequence of Phatr3_EG01992 was searched to obtain the nucleotide sequence encoded by Phatr3_EG01992 as shown in SEQ ID NO.5. Forward and reverse amplification primers containing KpnI restriction site were designed based on the Phatr3_EG01992 coding sequence. The nucleotide sequence of the forward amplification primer of the Phatr3_EG01992 coding sequence is shown in SEQ ID NO.6: 5'-GACTAATTCGAGCTCGGTACCATGAGGCTACGTTCATCCATTACC-3', and the nucleotide sequence of the reverse amplification primer of the Phatr3_EG01992 coding sequence is shown in SEQ ID NO.7: 5'-TCTAGAGGATCCCCGGGTACCACGGCAAAAGATTCCAAAACG-3';

[0052] The PCR amplification system was as follows: 0.5 μL of Phaeodactylum tricornutum DNA, 10 μL of 2× Phanta Flash Master Mix, 0.5 μL each of the forward and reverse amplification primers for the Phatr3_EG01992 coding sequence, and 8.5 μL of ddH2O;

[0053] The PCR amplification program was as follows: 94°C for 5 min; 94°C for 30 sec, 68°C for 30 sec, and 72°C for 1 min, 35 cycles, and storage at 4°C;

[0054] Step 3, PCR amplification product identification: After amplification, the PCR products were separated and verified by agarose gel electrophoresis. Figure 3 As shown, lane 1 is a DNA marker, and lanes 2 and 3 are the amplified Phatr3_EG01992 coding regions, which were then amplified and sequenced. The length of the amplified fragments was consistent with the theoretical length.

[0055] Step 4, construction of directional expression vector: The pPha-J47869-eGFP plasmid was digested with KpnI restriction endonuclease, and the amplified product of the Phatr3_EG01992 coding sequence obtained in step 3 was ligated with the digested pPha-J47869-eGFP. The reaction was incubated at 37°C for 30 min to construct the directional expression plasmid pPha-J47869-EG01992-eGFP.

[0056] Specific Example 3: Construction of enabling plasmid pPha-J47869-EG01992-eGFP-FSP.

[0057] The amino acid sequence of the FSP short peptide is shown in SEQ ID NO.8: MHRSDLMSAAVR, which has an efficient and specific affinity for silica. If the short peptide is directed to be expressed on the outside of the microalgae cells, it can give the microalgae cells the ability to efficiently bind to silica. Since the density of silica is much greater than that of water, the microalgae cells bound to silica can quickly settle to the bottom of the water. The microalgae germplasm transformed by this strategy can use silica as a flocculant to construct an efficient flocculation system, providing a new solution for the collection of microalgae cells. The steps for constructing the enabling plasmid pPha-J47869-EG01992-eGFP-FSP are as follows:

[0058] Step 1, synthesizing a double-stranded template: The coding sequence of the FSP short peptide was codon-optimized to obtain a nucleotide sequence encoding FSP as shown in SEQ ID NO.9: 5'-ATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3', and FSP double-stranded forward and reverse synthesis primers were designed based on the FSP coding sequence and the flexible linker (the nucleotide sequence of the flexible linker is shown in SEQ ID NO.18: GGGGSGGGGS) coding sequence, and the two synthesis primers were synthesized into double-stranded nucleotides as a PCR amplification template by annealing reaction, wherein the nucleotide sequence of the FSP double-stranded forward synthesis primer is shown in SEQ ID NO.10: 5'-GGCGGCGGTGGTAGCGGTGGCGGCGGTTCCATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3', and the nucleotide sequence of the FSP double-stranded reverse synthesis primer is shown in SEQ ID NO. Shown in NO.11: 5'-TTACCGCACAGCAGCCGACATGAGGTCGCTGCGGTGCATGGAACCGCCGCCACCGCTACCACCGCCGCC-3';

[0059] Step 2, designing FSP forward and reverse amplification primers based on the XbaI restriction site sequence and the FSP coding sequence, wherein the nucleotide sequence of the FSP forward amplification primer is shown in SEQ ID NO.12: 5'-GACGAGCTGTACAAGTCTAGGGCGGCGGTGGTAGCGGT-3', and the nucleotide sequence of the FSP reverse amplification primer is shown in SEQ ID NO.13: 5'-TGCCTGCAGGTCGACTCTAGATTACCGCACAGCAGCCGA-3';

[0060] The PCR amplification system was as follows: 0.5 μL double-stranded nucleotide, 10 μL 2× PrimeSTAR Max Premix, 0.5 μL each of the forward and reverse amplification primers for the FSP gene, and 8.5 μL ddH2O;

[0061] The PCR program was as follows: 94°C for 5 min; 94°C for 30 sec, 62°C for 30 sec, 72°C for 1 min, 35 cycles, and storage at 4°C.

[0062] Step 3, PCR amplification product verification: After amplification, the PCR products were separated by agarose gel electrophoresis, followed by gel excision, recovery and sequencing. The sequencing results were consistent with the FSP theoretical coding sequence;

[0063] Step 4, enabling plasmid construction: The pPha-J47869-EG01992-eGFP plasmid was digested with XbaI restriction enzyme, and the FSP amplification product obtained in step 3 was ligated with the digested pPha-J47869-EG01992-eGFP. The ligation was carried out at 37°C for 30 min to construct the enabling plasmid pPha-J47869-EG01992-eGFP-FSP, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0064] Specific Example 4: The enabling plasmid pPha-J47869-EG01992-eGFP-FSP constructed in Specific Example 3 was electroporated into Phaeodactylum tricornutum. The specific steps are as follows:

[0065] The pPha-J47869-EG01992-eGFP-FSP plasmid constructed in Example 3 was transformed into Phaeodactylum tricornutum by electroporation. The transformed Phaeodactylum tricornutum cells were plated on f / 2 plate culture medium, and positive algal strains were screened and identified to obtain genetically engineered algae with controllable flocculation. The specific steps were as follows:

[0066] Step 1, transformation of Phaeodactylum tricornutum: The enabling plasmid pPha-J47869-EG01992-eGFP-FSP was transformed into competent cells. After incubation, the cells were spread onto LB solid medium. After visible colonies were formed, the colonies were picked and transferred to LB liquid medium. The transformed competent cells were cultured in a constant temperature shaking incubator at 37°C. The transformed competent cells were collected, the pPha-J47869-EG01992-eGFP-FSP plasmid was extracted, and the cells were transformed into Phaeodactylum tricornutum by electroporation. The electroporation parameters were: 500 V, 25 μF, 400 Ω;

[0067] Step 2, screening of positive transformants: The transformed P. tricornutum cells were spread on f / 2 plate medium containing 1.0% agar with a bleomycin concentration of 75 μg / mL and placed under a light intensity of 60 μmol photons m -2 s -1 After about 3 weeks, the successfully transformed strains formed algal colonies on the f / 2 plates. Figure 4 As shown in the figure; visible algal colonies were picked and cultured in liquid f / 2 medium with a bleomycin concentration of 75 μg / mL for 1 week to obtain the algal liquid of P. tricornutum, and the DNA of the algal liquid was extracted as an identification template;

[0068] Step 3, identification of positive transformants: Identification primers were designed based on the eGFP coding sequence. The nucleotide sequence of the algae liquid forward identification primer is shown in SEQ ID NO. 14: 5'-GACGACGGCAACTACAAGAC-3', and the nucleotide sequence of the algae liquid reverse identification primer is shown in SEQ ID NO. 15: 5'-CGAACTCCAGCAGGACCAT-3';

[0069] The PCR amplification system was as follows: 0.5 μL of Phaeodactylum tricornutum DNA, 10 μL of 2× Phanta Flash Master Mix, 0.5 μL each of the forward and reverse identification primers designed based on the eGFP coding sequence, and 8.5 μL of ddH2O.

[0070] The PCR amplification program was as follows: 94°C for 5 min; 94°C for 30 sec, 58°C for 30 sec, 72°C for 1 min, 35 cycles, and stored at 4°C. PCR products were separated by agarose gel electrophoresis and then sequenced. Figure 5 As shown, the Sanger sequencing results showed that the amplified fragment originated from the eGFP coding region of the enabling plasmid, indicating that the enabling plasmid had been successfully transformed into P. tricornutum.

[0071] Specific Example 5: Verification of positive transformants of Phaeodactylum tricornutum.

[0072] 1. Verification of transcriptional level: Total RNA was extracted from each strain of P. tricornutum and reverse transcribed to obtain cDNA. Using cDNA as a template, the transcription of Phatr3_EG01992 in wild strains and positive transformants was compared and analyzed by real-time quantitative PCR.

[0073] Real-time quantitative PCR forward and reverse primers were designed based on the Phatr3_EG01992 sequence. The nucleotide sequence of the real-time quantitative PCR forward primer is shown in SEQ ID NO. 16: 5'-CGAGAACCGAAGTGACCG-3'; the nucleotide sequence of the real-time quantitative PCR reverse primer is shown in SEQ ID NO. 17: 5'-ACCAGCACAACCAGGGAC-3';

[0074] Real-time quantitative PCR system: 0.5 μL of Phaeodactylum tricornutum cDNA, 10 μL of 2× PhantaQuantiNovaSYBR Green PCR Master Mix, 0.5 μL each of the forward and reverse primers for real-time quantitative PCR, and 8.5 μL of ddH2O;

[0075] The real-time quantitative PCR program was as follows: 94°C for 5 min; 94°C for 15 sec, 60°C for 30 sec, 40 cycles. Figure 6 As shown, in the transformant transformed with the enabling plasmid, the transcription abundance of Phatr3_EG01992 was several times higher than that of the wild type (negative control 1) and the transformant transformed with the inducible expression plasmid pPha-J47869-eGFP (negative control 2), indicating that the target nucleotide sequence was successfully transcribed in the transformant transformed with the enabling plasmid.

[0076] 2. Protein level verification: Each strain of P. tricornutum was cultured to the logarithmic growth phase (cell density was about 5×10 6 cells / mL), the position and intensity of the eGFP signal of each strain of P. tricornutum cells were detected and compared using a laser confocal microscope at excitation and emission wavelengths of 488 nm and 507 nm, respectively. Figure 7 As shown, the green fluorescence signal of the P. tricornutum strain transformed with the enabling plasmid was accurately located on the outside of the cell; no eGFP signal was observed in the cells of the wild strain (negative control 1); while the eGFP signal of the strain transformed with the inducible expression plasmid pPha-J47869-eGFP (negative control 2) was located in the cytoplasm.

[0077] In summary, after identification of transcription and protein expression levels, it can be determined that the FSP-enabling factor carried by the positive transformant can be correctly transcribed, translated, and accurately localized to the outside of the P. tricornutum cells.

[0078] Specific Example 6: Verification of the efficient flocculation effect based on the positive transformant strain of Phaeodactylum tricornutum.

[0079] Cultivate P. tricornutum: The positive transformant strain of P. tricornutum and the wild strain (negative control) were inoculated into liquid f / 2 culture medium at the same starting density (cell density was about 1×10 6 cells / mL) and cultured in a constant temperature and light shaker; the culture conditions were a light intensity of 60 µmol photons m -2 s -1 , temperature 22℃, speed 90 rpm.

[0080] Culture to the plateau phase (cell density is about 4×10 7 After culturing the algae in a constant temperature and light shaker for 24 hours, the algae solution was left to stand for 5 minutes. The results were as follows: Figure 8 As shown, the positive transformed strains of P. tricornutum will quickly settle to the bottom of the culture container due to the binding of a large amount of silica, achieving efficient and low-energy collection; however, the wild strain of P. tricornutum cells lacks the ability to bind silica, and its cells are still evenly distributed in the algae liquid and cannot be collected.

[0081] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. A genetically engineered algae capable of controlled flocculation, characterized by: The genetically engineered algae is Phaeodactylum tricornutum containing an enabling plasmid pPha-J47869-EG01992-eGFP-FSP. The FSP is directionally expressed outside the cells of Phaeodactylum tricornutum. The nucleotide sequence of the enabling plasmid pPha-J47869-EG01992-eGFP-FSP is shown in SEQ ID NO.

1.

2. A method for constructing a genetically engineered algae capable of controlled flocculation according to claim 1, characterized in that The following steps are involved: Step 1: Using the genomic DNA of Phaeodactylum tricornutum as a template, the Phatr3_J47869 promoter was amplified and replaced with the FcpA promoter of the basic expression plasmid pPha-T1-eGFP to construct the inducible expression plasmid pPha-J47869-eGFP; Step 2: Using the genomic DNA of Phaeodactylum tricornutum as a template, the coding sequence of Phatr3_EG01992 was amplified and inserted upstream of the eGFP of the inducible expression vector to construct the directional expression plasmid pPha-J47869-EG01992-eGFP; Step 3: artificially synthesize the codon-optimized FSP coding sequence and insert it into the downstream of eGFP in the directional expression vector to construct the enabling plasmid pPha-J47869-EG01992-eGFP-FSP; Step 4: electro-transform the enabling plasmid pPha-J47869-EG01992-eGFP-FSP into Phaeodactylum tricornutum, and spread the transformed Phaeodactylum tricornutum cells on f / 2 plate culture medium for culture, and screen positive algae strains to obtain genetically engineered algae with controllable flocculation.

3. The method for constructing a genetically engineered algae capable of controlled flocculation according to claim 2, characterized in that Step 1 is as follows: (1) Based on the Phatr3_J47869 promoter sequence, a forward amplification primer containing an NdeI restriction site and a reverse amplification primer containing an EcoRI restriction site were designed. PCR amplification was performed using P. tricornutum DNA as a template to obtain the Phatr3_J47869 promoter amplification product. The nucleotide sequence of the Phatr3_J47869 promoter forward amplification primer is shown in SEQ ID NO.3: 5'-GTACTGAGAGTGCACCATATGTGGTGGTGAATCAACACTTAATGTG-3', and the nucleotide sequence of the Phatr3_J47869 promoter reverse amplification primer is shown in SEQ ID NO.4: 5'-TTAGTCGATGATATCGAATTCCGAAGAATTCTTTTCACCAGGG-3'; (2) The original FcpA promoter of pPha-T1-eGFP was removed using NdeI and EcoRI restriction endonucleases; the amplified product of the Phatr3_J47869 promoter was connected with the enzyme-cleaved product of pPha-T1-eGFP to construct the inducible expression plasmid pPha-J47869-eGFP.

4. The method for constructing a genetically engineered algae capable of controlled flocculation according to claim 2, characterized in that Step 2 is as follows: (1) Forward and reverse primers containing KpnI restriction sites were designed based on the coding sequence of Phatr3_EG01992. PCR amplification was performed using the DNA of Phaeodactylum tricornutum as a template to obtain the amplified product of the coding sequence of Phatr3_EG01992. The nucleotide sequence of the primer for forward amplification of the Phatr3_EG01992 coding sequence is shown in SEQ ID NO. 6: 5′-GACTAATTCGAGCTCGGTACCATGAGGCTACGTTCATCCATTACC-3′, and the nucleotide sequence of the primer for reverse amplification of the Phatr3_EG01992 coding sequence is shown in SEQ ID NO. 7: 5′-TCTAGAGGATCCCCGGGTACCACGGCAAAAGATTCCAAAACG-3′; (2) The pPha-J47869-eGFP plasmid was digested with KpnI restriction endonuclease, and the amplified product of the Phatr3_EG01992 coding sequence was ligated with the digested pPha-J47869-eGFP to construct the directional expression plasmid pPha-J47869-EG01992-eGFP.

5. The method for constructing a genetically engineered algae capable of controlled flocculation according to claim 2, characterized in that Step 3 is as follows: (1) Synthesis of double-stranded template: The coding sequence of the FSP short peptide was codon-optimized to obtain the nucleotide sequence of the FSP coding gene as shown in SEQ ID NO.9: 5'-ATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3'. FSP double-stranded forward and reverse synthesis primers were designed based on the FSP coding sequence and the flexible linker coding sequence. The two synthesis primers were synthesized into double-stranded nucleotides as PCR amplification templates by annealing reaction. The nucleotide sequence of the FSP double-stranded forward synthesis primer was shown in SEQ ID NO.10: 5'-GGCGGCGGTGGTAGCGGTGGCGGCGGTTCCATGCACCGCAGCGACCTCATGTCGGCTGCTGTGCGGTAA-3', and the nucleotide sequence of the FSP double-stranded reverse synthesis primer was shown in SEQ ID NO.11: 5'-TTACCGCACAGCAGCCGACATGAGGTCGCTGCGGTGCATGGAACCGCCGCCACCGCTACCACCGCCGCC-3'. (2) Design FSP forward and reverse amplification primers based on the XbaI restriction site sequence and the FSP coding sequence, and use the double-stranded nucleotide obtained in step (1) as a template for PCR amplification to obtain an amplification product containing the FSP coding sequence, the corresponding restriction site and the linker coding sequence, wherein the nucleotide sequence of the FSP forward amplification primer is shown in SEQ ID NO.12: 5'-GACGAGCTGTACAAGTCTAGGGCGGCGGTGGTAGCGGT-3', and the nucleotide sequence of the FSP reverse amplification primer is shown in SEQ ID NO.13: 5'-TGCCTGCAGGTCGACTCTAGATTACCGCACAGCAGCCGA-3'; (3) The pPha-J47869-EG01992-eGFP plasmid was digested with XbaI restriction endonuclease, and the FSP amplification product was ligated with the digested pPha-J47869-EG01992-eGFP to construct the enabling plasmid pPha-J47869-EG01992-eGFP-FSP, the nucleotide sequence of which is shown in SEQ ID NO.

1.

6. The method for constructing a genetically engineered algae capable of controlled flocculation according to claim 2, characterized in that Step 4 is as follows: (1) Transformation of Phaeodactylum tricornutum: The enabling plasmid pPha-J47869-EG01992-eGFP-FSP was transformed into competent cells, and after incubation, the cells were spread onto LB solid culture medium; after visible colonies were formed, the colonies were picked and transferred to LB liquid culture medium for shaking culture; the transformed competent cells were collected, the enabling plasmid pPha-J47869-EG01992-eGFP-FSP was extracted, and the cells were transformed into Phaeodactylum tricornutum by electroporation; (2) Screening of positive transformants: The transformed P. tricornutum cells were spread on a solid f / 2 plate medium containing bleomycin and placed under a light intensity of 50-70 μmol photons m -2 s -1 , culture under the conditions of temperature of 20-24℃, screen the positive transformation columns, and obtain the genetically engineered algae with controllable flocculation.

7. Use of the controllable flocculation genetically engineered algae according to claim 1 in preparing a microalgae flocculation system, characterized in that The microalgae flocculation system is an algae liquid added with white carbon black, and the amount of the white carbon black added is 0.5-1.5 g per liter of the culture liquid of the triangular algae.

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