Experiments

Algal species

     Phaeodactylum tricornutum（P. tricornutum） is provided by the CCMP algae repository in the United States, under the species label CCMP2561.

Algal culture

     The marine diatoms P. tricornutum were grown in batches in triangular conical flasks containing f/2-Si medium and incubated in an artificial climate chamber. The parameters of the artificial climate chamber were: temperature 21 ± 1°C, photoperiod 12 h/12 h (L/D), and light intensity 150 μmol photon m^-2 s^-1.

     The 100 mL of algal solution was incubated in 250 mL triangular conical flasks and the mouths of the flasks were sealed with a sealing film containing 0.22 μm filter membrane to isolate airborne bacteria and other materials. The algal solution was manually shaken three times a day during the incubation period to slow down the aggregation of cells settling in the triangular conical flask.

Bacteria species

     Qipengyuania aquimaris is provided by MCCC (Marine Culture Collection of China), under the species label MCCC 1A14896.

Bacteria culture

     The marine bacteria Qipengyuania aquimaris were grown in triangular conical flasks containing 2216e culture medium, incubated in an artificial climate chamber. The parameters of the artificial climate chamber were: temperature 21 ± 1°C, photoperiod 12 h/12 h (L/D), and light intensity 150 μmol photon m^-2 s^-1.

     The 100 mL of bacteria solution was incubated in 250 mL triangular conical flasks and the mouths of the flasks were sealed with a sealing film containing 0.22 μm filter membrane to isolate airborne bacteria and other materials. The bacteria solution was manually shaken three times a day during the incubation period to slow down the aggregation of cells settling in the triangular conical flask.

Interactions between microalgae and bacteria

     For the ratio of microalgae to bacterial cell density in culture, we set up six experimental groups: R1(microalgae: bacteria = 1:0, microalgae only) , R2(microalgae: Bacteria = 1:1) , R3(microalgae: Bacteria = 1:3) , R4(microalgae: bacteria = 1:5) , R5(microalgae: bacteria = 1:7) , R6(microalgae: bacteria = 0:1, bacteria only) .

Microalgae & Bacteria cell count

     The cell density of P. tricornutum was observed and counted by hemocytometer plate and light microscope analysis.

     The density formula was CD = (N/80) × 400 × 10^4, where N is the total number of cells in 80 small compartments of the hemocytometer plate.

    The optical density of Phaeodactylum tricornutum was measured at the wavelength of 680 nm, and the optical density of bacteria was measured at 600 nm.

Microalgae & Bacteria growth curve determination

     At least 400 cells were counted by light microscopy with a hemocytometer plate and cell growth curves were plotted for each treatment group at regular intervals daily during the Phaeodactylum tricornutum triangle culture cycle.

     Enrichment of 500 ml of algal or bacteria solution was performed using a sterile 50 ml centrifuge tube at 4400 rpm, 4°C for 10 min. The supernatant was discarded, the precipitate was rinsed with RO water, the supernatant was discarded by centrifugation, and the procedure was repeated three times. The collected algal precipitate was transferred to 1.5 ml EP tubes and snap-frozen in liquid nitrogen, followed by freeze-drying for 12 h or longer until all water evaporated. Finally, after snap-freezing in liquid nitrogen, the algal precipitate was immediately stored at -80°C.

     Liquid nitrogen is poured into the mortar to pre-cool the mortar and mortar rod. The enriched algal or bacteria masses were then poured into the mortar and quickly ground to a powder, while liquid nitrogen was continuously added to ensure that the mortar and sample were kept at a low temperature. Finally, the powder was poured into 1.5 ml RNAse free EP tubes and placed back into liquid nitrogen for freezing. The powder was freeze-dried in a freeze-dryer until all the water was evaporated, then frozen in liquid nitrogen and stored at -80°C.

Reagents:

1. Digestant solution: 20g of potassium persulfate (K₂S₂O₈ 2times recrystallized) and 3g of sodium hydroxide (NaOH) dissolved in water and diluted to 1000ml.
2. Nitrogen standard storage solution: weigh 0.7218g of potassium nitrate (KNO₃) dissolved in water and diluted to 1000ml. This solution is 100ug/ml of nitrate nitrogen (NO₃-N).
3. Phosphorus standard stock solution: weigh 0.4394g of potassium dihydrogen phosphate (KH₂PO₄) baked at 45℃ for 4-8 hours, dissolved in water and add 1ml (1 + 1) H₂SO₄ solution and diluted to 1000ml. This solution is 100ug/ml of phosphorus stock solution.
4. Molybdenum-antimony storage solution of N sulfuric acid: Take 180.6 ml of concentrated sulfuric acid (H₂SO₄) and add it slowly to 400 ml of water with constant stirring and cool. Also weigh ammonium molybdate [(NH₄)2MoO₄-4H₂O] 20g Dissolve in 300 ml of water at approx. 60°C, stir constantly, add 100 ml of 0.5% antimony potassium tartrate solution, cool and dilute to 1000 ml with distilled water, shake well and store in brown reagent bottles.
5. Molybdenum-antimony anti-mixed color developer: In 100 ml of molybdenum-antimony storage solution, add 1.5 g of L-ascorbic acid.

    This reagent is effective 24 hours, should be prepared before use.

Steps:

1. Add a reagent to the nitrogen-phosphorus glass tube.
2. Sample digestion: glass tube cover and tighten the lid, wrapped with a sealing film, into the sterilizer 121 ℃, digestion for 30 min; digestion is complete, timely shaking of nitrogen-phosphorus glass tube in the liquid to avoid precipitation undissolved, remove the sealing film, and wait for the liquid to cool to room temperature.
3. Determination by spectrophotometry: Use Nanodrop, select the ultraviolet spectrophotometry program (UV-Vis), uncheck the Baseline correction when measuring, use the wavelengths of 220 nm and 276 nm for nitrogen measurement, and use the wavelength of 700 nm for phosphorus measurement, and draw up 2.5 μL of the sample each time to carry out the above measurements, and then draw a standard curve according to the measured data. The standard curve was plotted based on the measured data.

Fucoxanthin standard curve

    Take 11 test tubes, number them, add various solutions according to Table 1, and then measure the absorbance value at 445nm with the mixture of test tube No.0 as reference. Taking fucoxanthin as abscissa and absorbance as ordinate, the regression equation was obtained.

Determination of fucoxanthin content

    The extraction and content determination of fucoxanthin from P.tricornutum were carried out by organic solvent extraction method. 80mL of P. tricornutumwas accurately extracted and centrifuged at 5000r·min^-1，4℃ for 10min. The supernatant was discarded, freeze-dried for 2d, weighed and ground into powder about 0.1g with anhydrous ethanol, alga powder: anhydrous ethanol =1:40(g·mL1),60 The supernatant was taken and the absorbance was measured at 445nm wavelength (OD445) by uv spectrophotometer. The content of fucoxanthin was calculated by regression equation

1. On the 4th, 6th, 8th and 10th day of cultivation, 2 mL algae solution was collected in the EP tube weighed in advance, frozen and vacuum dried for 12 h, and the corresponding algae weight was obtained by weighing.
2. Each tube was added with 1.5 mL of 95 % ethanol at 4 °C for 24 h in the dark with periodic oscillation during soaking.
3. After the extraction was completed, the supernatant was separated by centrifugation at 4℃, 12000 rpm for 10 min, and the supernatant was injected into the microplate. The absorbance at 470 nm, 649 nm and 665 nm was read and the chlorophyll concentration was calculated according to the formula to calculate the pigment content.

C_chla (mg·L^-1)= 13.95A₆₆₅ – 6.88A₆₄₉

C_chlb (mg·L^-1)= 24.96A₆₄₉ – 7.32A₆₆₅

C_carotenoid (mg·L^-1)=(1000A₄₇₀ – 2.05 C_chla -114 C_chlb)/245

pigment content (mg·g-1DW) = pigment concentration × extraction volume（L）/ sample dry weight（g）

    Total carbohydrate content was quantified using the phenol-sulphuric acid method . About 10 mg of lyophilized algae was resuspended in 1 mL of deionized water. This algal suspension was added with 1 mL of 5% phenol solution (w/v) was rapidly added directly to be mixture. The mixture was incubated for 10 min at room temperature and then 30 °C for 20 min. The content of carbohydrates was detected at 483 nm. Glucose was used as standard at different concentrations (up to 1 g·L⁻¹).

     Total lipid content was also determined by gravimetric analysis. About 20 mg of lyophilized algae was mixed with 2 mL of chloroform, 2 mL of methanol and 1 mL of 5% NaCl by vortex for 2 min, then centrifuged at 8000g for 4 min at 10 °C. The chloroform phase was collected and kept for subsequent analysis. The residual extract was extracted three more times. All the collected chloroform phase were mixed together and dried under nitrogen flow. The dried lipid residue was further dried in oven at 60 °C and the total lipid content was quantified as a percentage of the dried weight of algae.

1. Take appropriate amount of algae liquid (the same group is recommended to do 5 groups of parallel)
2. Add 15% volume fraction of DMSO, add Nile red dye; dark 20min, during which 3-5min vortex shock increases dyeing efficiency.
3. The 300ul sample was added to the enzyme plate, and the same volume of PBS was used as the blank control.
4. Parameters detected by enzyme labeling instrument: Read Mode: RFU; Wavelengths: Wavelengths 1 Magneto ExcitationRod 488nm Emission:592nm, Auto Cutoff

    To visualize oil bodies and assess their morphology, localization, and numbers in the cultured cells, Nile red and BODIPY 505/515 were applied under dark conditions in accordance with a similar procedure to that described for the determination of neutral lipid and TAGs contents. Mixtures were thoroughly mixed, applied to a glass slide, covered with a coverslip after 10 min, and then observed under a Laser scanning confocal microscope LSM (Leica) (Nile Red: excitation 543 nm, emission 570–610 nm; BODIPY 505/515: excitation 488 nm, emission 530 nm). Pictures were randomly acquired from at least 20 cells per sample, and typical images are presented.

1. Microalgae in logarithmic growth phase ( the fastest growing ) were inoculated in a column photobioreactor, 1 % CO₂,23 ° C, 300 μmol photons / m² · s continuous illumination for 24 h.
2. 2.5 ml of microalgae solution containing about 2 × 106 microalgae cells was pretreated by microwave oven for 40 s, 1 % dimethyl sulfoxide ( DMSO ) was added, 75 μl 50 μg / ml NR ( final concentration was 1.5 μg / ml ) was added, 40 ° C was dark, and Nile red ( NR ) dye was used for 5 min.
3. 300 × g centrifuged at room temperature for 5 min, discard the supernatant.
4. Add 4 ml PBS to the precipitation of microalgae cells, mix with a pipette gun, centrifuge at 300 × g for 5 min at room temperature, and discard the supernatant.
5. The microalgae cells were resuspended with 2 ml PBS, washed, and centrifuged at 300 × g at room temperature for 5 min. The supernatant was carefully removed and the cell precipitate was collected. Repeat once.
6. The excess fluorescent dye was removed, and 200 μl liquid was reserved, which was filtered by 70 μm filter membrane and detected by flow cytometry.
7. On a flow cytometer, the detection parameters ( NR excitation wavelength of 543 nm, emission wavelength of 598 nm, fluorescence signal located in FL3 channel ) were first set with unstained microalgae cell suspension, and the negative region was adjusted. At least 1 × 105 positive cells were obtained for subsequent data analysis.

     By using AquaPen AP110, the QY, OJIP and NPQ1 indexes of algae liquid were measured, which reflected the energy conversion efficiency of Phaeodactylum tricornutum in photochemical activity.

     The concentration of 1 μl sample was determined in the DNA mode of ThermoTM NanoDrop2000/200C.

     Agarose was dissolved into 1X TAE solution (1.2% or 1.5%, i.e. 1.2g/100ml or 1.5g/100ml) using an electronic analytical balance, heated in microwave oven to dissolve, waited for the temperature to drop to about 50~60℃, poured into the gel version, and pulled off the mold for sampling after the agarose gel solidified, and selected a suitable program for electrophoresis.

1. In a 1.5 ml microcentrifuge tube, add 5 volumes of Binding Buffer to 1 volume of PCR products (50-100 μl). Mix briefly by vortexing sample.
2. Transfer all the mixture to a provided Spin Column with a Collection Tube (to increase the yield of purified DNA, incubate for 1 minute).
3. Centrifuge at 10,000 × g for 1 minute. Discard the flow-through.
4. Add 650 μl of Wash Buffer to the column. Centrifuge at 10,000 × g for 1 minute. Discard the flow-through.
5. Centrifuge the empty column at 10,000× g for 1-2 minutes to remove any residual Wash Buffer.
6. Place the spin column in a clean microcentrifuge tube, add 30-50 μl of Elution Buffer or sterile, distilled water (pH >7.0) directly to the center of the column matrix (to increase the yield, prewarmed Elution Buffer or water can be used). Incubate the column at room temperature for 1 minute. Centrifuge at 10,000xg for 1minute to elute the DNA. The isolated DNA is ready to use or can be stored at -20°C.

1. Centrifuge overnight bacterial cell culture (refer to above table) at 12000×g for 2 minutes and discard the supernatant.
2. Add 5/10 ml of colorless Resuspension Buffer (containing RNase A). Mix thoroughly by vortexing.
3. Add 5/10 ml of blue Lysis Buffer, mix by gently inverting the tube 4-6 times to completely lyse cells. Incubate at room temperature for 5 minutes (lysate should change color to blue).
4. Add 7/14 ml of yellow Neutralization Buffer, mix by gently inverting the tube 5-6 times. Incubate at room temperature for 2 minutes (lysate should change color to yellow).
5. Centrifuge at 12,000×g for 15 minutes, gently transfer the supernatant to a spin column(if more than 50 ml, repeat the transfer after step 6).
6. Centrifuge at 8000×g for 2 minutes and discard the flow-through.
7. Add 5 ml of ToxinOut Buffer, incubate at room temperature for 10 minutes. Centrifuge at 8,000×g for 2 minutes and discard the flow-through.
8. Add 3/5 ml of Wash Buffer, centrifuge at 8,000×g for 2 minutes and discard the flow-through.
9. Repeat step 8 once.
10. Centrifuge at 8,000×g for 5 minutes to completely remove the remaining Wash Buffer.
11. Incubate at room temperature for 10 minutes to evaporate ethanol.
12. Place the spin column in a clean 50 ml centrifuge tube, add 1-2 ml of of EB or sterile, distilled water (pH>7.0) directly to the center of the column matrix (for higher yield, use prewarmed (60-70℃) Elution Buffer or distilled water).
13. Centrifuge the column at 8000×g for 2 minutes to elute DNA.
14. Isolated plasmid DNA can be stored at -20℃.

1. Thaw a vial of Trans1-T1 Phage Resistant Chemically Competent Cell on ice.
2. Transfer 2 μl of reaction mixture into 50 μl of Trans1-T1 Phage Resistant Chemically Competent Cell and mix gently by flicking the tube (do not vortex). Incubate on ice for 30 minutes.
3. Heat-shock in a water bath at 42℃ for 30 seconds, and immediately place on ice for 2 minutes.
4. Add 450 μl of room-temperature SOC/LB medium. Place in a shaking incubator at 37℃ for 1 hour at 250 rpm.
5. Pre-warm LB plate containing the appropriate selection antibiotic at 37℃.
6. Spread 100 μl of cells evenly on the selection plate and incubate overnight at 37℃.

1. Add the following components in a pcr tube:

2. Perform PCR with thermocycle:

1. Add the following components in a pcr tube:

2. Perform PCR with thermocycle:

     FlyCut" Sacl is expressed and purified from E.coli that carries the recombinant Sacl gene. The molecular weight is 40.8 kDa, with the recognition site at GAGCT^C. The reaction is conducted for 5- 15 minutes at 37°C, and heat-inactivated at 80℃ for 20 minutes. This enzyme is not sensitive to dam, dcm or mammalian CpG methylation.

     FlyCut Xhol is expressed and purified from E.coli that carries the recombinant Xhol gene. The molecular weight is27.9 kDa, with the recognition site at C^TCGAG. The reaction is conducted for 5- 15 minutes at 37°C, and heat-inactivated at 65°℃ for 20 minutes. This enzyme is not sensitive to dam or dcm methylation, but sensitive to mammalian CpG methylation.

1. Equilibrate a water bath to 42℃.
2. Warm a vial of SOC medium or LB medium to room temperature. Warm selective plates at 37°C for 30 minutes.
3. Thaw a vial of 100 μl of Trans1-T1 Phage Resistant Chemically Competent Cell on ice, aliquot 50 μl of the cells into a prechilled
4. 1.5 ml tube, add target DNA (1 to 5 μl) into the tube. Do not mix by pipetting up and down. Incubate the cells on ice for 30 minutes.
5. Heat-shock the cells for 30 seconds at 42°C without shaking. Immediately transfer the tube to ice. 6.Incubate on ice for 2 minutes without shaking.
6. Add 500 μl of prewarmed SOC medium or LB medium (without antibiotic) into the tube, mix well and shake at 37℃ for 1 hour at 200 rpm for cell recovery and for the expression of antibiotic resistance.
7. Spread 20 to 200 μl from each transformation vial on a prewarmed selective plate. The remaining can be stored at 2-8℃ and plated the next day if needed.
8. Invert the plate and incubate at 37°C overnight.
9. Select colonies and analyze by restriction enzyme digestion, PCR, or sequencing.

     To further validate the RNA-Seq results, RNA extracted from the same culture for RNA-Seq was subjected to the PrimeScript RT reagent kit with gDNA Eraser for cDNA synthesis. qPCR was performed using standard methods as previously described . β-Actin was used as a housekeeping marker. Primer pairs used for qPCR analysis are listed in attachment.

1. Leach the algae ball and freeze it with liquid nitrogen, grinding into powder, packed in 1.5 ml EP tube.
2. Add 1 ~ 2.5 times of NG buffer to the fume hood, and mix well on the vortex meter.
3. Heating on a 100 °C thermostat for 10 min.
4. 12,000 rpm Centrifugation for 10 min.
5. Take the supernatant for SDS-PAGE electrophoresis （step1：86V， 30min；step2：130V，1.5h）.

1. After the electrophoresis, cut off the protein gel block, place it in a protein semi-dry transfer instrument, and transfer the target protein to the PVDF membrane under the condition of 15 V 13 min.
2. Put the PVDF membrane into 50 g/L skimmed milk powdr shake).
3. Use 20 g/L skimmed milk powder to dilute the anti-His mouse antibody at a ratio of 12000, and add an appropriate amount of the diluted antibody.
4. Submerge the PVDF membrane in a self-made closed bag containing PVDF membrane, and incubate overnight at 4 'C (slowly shaking).
5. Take out the PVDF mem-brane from the primary antibody solution and place it in a petri dish, wash with TBST 5 times, each time 15min.
6. Use 20 g/L skimmed milk powder to dilute goat anti-mouse lgG (called secondary antibody) at a ratio of 1:3000, and add an appropriate amount of diluted secondary antibody to a self-made closed bag with PVDF membrane to submerge the PVDF membrane and incubated at room temperature for 2 hours(slowly shake).
7. take out the PVDF membrane from the secondary antibody solution and place it in a plate, wash five times with TBST, 15 minutes each time.
8. After washing. dry the PVDF membrane and drop it on the front. Add the prepared color developer until it is completely covered. place it at room temperature for 2 minutes, place it in an ECL luminescence imager for exposure and photographing. and record the result.