Experiments

LiDSI approach

Cloning of pTpNR_T8 Vector

The first step was to prepare the pTpNR_T8 vector. This was accomplished through an enzyme restriction digest, using EcoRI in 10x Fast Digest buffer, Milli Q water and the restriction enzyme EcoRI (10 U/µL) (parameters specified in the table below) following the manufacturer's protocol (New England Biolabs).

After the enzymatic digestion, the vector was incubated in a water bath, maintaining a temperature of 37°C for a duration of 3 hours. To verify the enzymatic digestion, the digested vector was subjected to 1.0% agarose gel electrophoresis. Following electrophoresis, the appropriate DNA fragment, measuring 5.4 kb, was excised from the gel (under UV-light, excitation wavelength of 365 nm). To isolate and purify the selected DNA fragment, a DNA extraction process was performed using the Zymo DNA Recovery Kit, adhering to the manufacturer's protocol. Concentration of the recovered DNA was measured by a Synergy H1 Hybrid Reader from BioTek. Prior to the cloning process, concentrations of all constructs intended for fusion with the pTpNR_T8 vector, which carries an eGFP sequence, were also determined using the Synergy H1 Hybrid Reader from BioTek.

To determine the annealing temperatures, the NEB Tm Calculator was employed. Specific PCR conditions for each construct, including annealing temperatures and cycling parameters, were selected and documented in the following table.

Each sample was subjected to electrophoresis on a 1.0% agarose gel. After electrophoresis, the DNA fragments of the appropriate size were excised and purified using the Zymo DNA Recovery Kit. DNA concentrations for each construct were determined using the Synergy H1 Hybrid Reader from BioTek.

The concentrations of the Gibson Assembly reactions were determined based on calculations performed using an provided excel sheet for Gibson Assembly (Kröger Lab). Subsequent to concentration determination, the Gibson Assembly reactions were incubated in a Thermo cycler at 50°C for a duration of 60 minutes. Following the incubation, the samples were stored on ice until they were ready for transformation into E. coli DH5 alpha cells.

Chemical Transformation into E. coli DH5 alpha cells

The Gibson Assembly reactions were briefly centrifuged. Competent E. coli DH5 alpha cells, prepared in advance (see experimental part in vitro), were thawed on ice for approximately five minutes. Gently flicking the tubes ensured uniform distribution of the cells. 50 μL of the thawed competent E. coli cells were combined with 2.5 μL of the Gibson Assembly reaction, ensuring thorough mixing while avoiding vortexing, which could potentially damage the cells. The mixture was then subjected to a heat-shock treatment, immersed in a 42°C water bath for 30 seconds. Following the heat-shock, the tubes were immediately returned to ice, allowing for a two minute incubation. To support the recovery of the transformed cells, 500 μL of room temperature SOC (Super Optimal Broth with Catabolite repression) medium was added to each transformation mixture. The transformed cells were incubated up to 60 minutes at 37°C with gentle shaking at 300 rpm. In parallel, LBamp (100 µg/mL Ampicillin) plates were pre-warmed to 37°C. For plating, 100 μL of the transformation mixture was spread onto one LBamp plate, while the remaining portion was evenly distributed onto a second LBamp plate using glass beads. In the case of the rest of the sample solution, cells were briefly centrifuged for one minute at 11,000 x g, followed by the removal of 350 μL of the supernatant. The remaining E. coli pellet was then resuspended in the remaining 100 μL. Finally, the plates were positioned upside down and placed in an incubator, maintaining a temperature of 37°C.

The day following the overnight incubation, colonies that had formed on the LBamp plates were selected for further analysis. This was done by using a sterile toothpick to pick eight suitable colonies for each construct. Each selected colony was then introduced into a 2 mL culture of LB Medium supplemented with 100 µg/mL Ampicillin (Amp). These cultures were placed in snap-cap tubes for growth at 37°C, with continuous shaking at 220 rpm. Prior to initiating plasmid isolation, a small aliquot from each E. coli culture was plated onto a fresh LB Amp plate, at approximately one-quarter of the plate's surface in terms of strain preservation.

Test Digestion of Gibson Assembly

To confirm the success of the Gibson Assembly reactions and the presence of the desired plasmids, a test digestion was performed. The components required for the digestion, including appropriate enzymes, buffers, and DNA samples, were mixed together. The digestion mixture was subsequently incubated at 37°C for a minimum of one hour. Following the digestion, the entire reaction mixture was loaded onto a 1.0% agarose gel for electrophoresis.

Plasmid Preparation for Sequencing

In preparation for sequencing analysis, the plasmids containing the cloned DNA constructs were isolated. The purified plasmids were then prepared for sequencing (40-100 ng/µL plasmid concentration), ensuring that the genetic information encoded within the constructs was accurately verified.

Preparation of Co-Transformation

Artificial Sea Water was prepared according to the following protocol:

Prior to the co-transformation, diatom agar plates (3% Agar stock, prepared with 2X Artificial Sea Water (ASW) medium) were prepared. These plates were created both with and without Zeocin or Nourseothricin, with regard to transformation and selection of Zeocin-resistant or Nourseothricin-resistant cells. For each construct or transformation, two plates were prepared containing ASW without Zeocin or Nourseothricin and ten plates with ASW supplemented with Zeocin or Nourseothricin (150 µg/mL). Zeocin and Nourseothricin plasmid (pTpfc/nat or pTpfcp/ble) preparation was performed prior to this step.

The equipment for the Gene gun (Biolistic PDS-1000/He Particle Delivery System - BioRAD) was sterilized by heating it in an oven at 300°C for a minimum of 10 hours. Thalassiosira pseudonana CCMP1335 diatom cells were grown until they reached a desired cell density of approximately 1·10⁶cells/mL at 18 °C with day-night cycle of light. To confirm this density, cell counts were performed with the TC20 Automated Cell Counter (Bio-RAD). For each transformation, 100 mL of cell culture was centrifuged for 10 minutes at 3200 rcf and 18°C to achieve around 1·10⁸ cells/mL. After centrifugation, the supernatant was removed using a syringe, noting that decanting was not straightforward due to the less stable pellet formation of the transformed cells. Additional centrifugation steps were conducted with lower volumes to yield approximately 250-400 µL of cells. Using a wire loop, the diatom cells were evenly spread on the surface of an agar plate, forming a 5.2 cm diameter circle in the center. To facilitate even drying, the agar plates were rotated every 5 minutes until the cells were dry and firmly adhered to the agar.

Preparation of Tungsten Particles

10 mg of tungsten particles were resuspended in 500 μL of 80% ethanol. Following resuspension, the suspension was centrifuged for 30 seconds. The resulting pellet was resuspended in 250 μL of ethanol. While vortexing, suspension was mixed for a duration of 1-2 minutes, followed by another 30 second centrifugation step. The pellet was washed three times, with each washing step involving 250 μL of sterile water. Following the last washing step, the particles were resuspended in 150 μL of H₂O and evenly divided into 50 μL aliquots.

Coating of Tungsten Particles with Plasmid DNA

For each 50 µL aliquot of tungsten particles, plasmid DNA was added to a final concentration of 5 µg for each the plasmid containing the POI (pTpNR_T8) and for the plasmid containing the antibiotic resistance (pTpfc/nat or pTpfcp/ble). The volume was adjusted by removing the appropriate volume of water out of the prepared tungsten particles. Along with the plasmid DNA, 50 µL of 2.5 M CaCl₂ and 20 µL of 0.1 M spermidine were added under constant vortexing for a duration of 3 minutes. After vortexing, a brief centrifugation step (3 seconds at high speed) was performed, and the supernatant was removed. The pellet was resuspended in 250 µL of ethanol, followed by another 1-minute vortexing. A second centrifugation step (3 seconds at high speed) was executed, and the supernatant was removed. The pellet was finally resuspended in 30 µL of ethanol.

Co-Transformation

After allowing the diatom cells to dry on the agar plate, the DNA-coated tungsten particles were transferred to a freshly ethanol cleaned macrocarrier in the center of the macrocarrier holder and air-dried for approximately 10 minutes. A rupture disk, pressurized to 1550 psi, was inserted into the rupture disk holder. The macrocarrier holder unit was assembled, with attention given to the placement of the stopping screen. The petri holder (target shelf) was placed in the appropriate slot (third from the bottom).

The Biolistic Gene gun (Biolistic PDS-1000/He Particle Delivery System - Bio-RAD) was configured accordingly, and the Petri dish was positioned on the clear shelf in position 2 below the macrocarrier holder. The diatom cells were shot with the aim of creating maximum vacuum pressure inside the chamber, approximately 28 psi. Following this, the cells were carefully scraped from the petri dish using 5 mL of ASW medium. The cells were then incubated overnight under constant illumination in 100 mL of fresh ASW medium.

Post-Transformation

Centrifugation was performed for 10 minutes at 3,200 rcf and at 18°C. The cells were resuspended in 3 mL, and the cell count was performed using a 1:100 dilution. An additional 3 mL of ASW was added to each sample, adjusting the cell density to approximately 3-4·10⁵ cells/mL, confirmed through a 1:100 dilution. Sufficient cells were centrifuged to plate 5·10⁶ cells/mL cells on each agar plate. The cells were then spread onto agar plates containing 150 μg/mL Zeocin or Nourseothricin. The plates were sealed with parafilm and incubated for six to ten days at 18°C under constant illumination, positioned upright.

No cell growth on Zeocin plates was detectable. Therefore, the co-transformation steps were repeated using a Nourseothricin (ClonNAT) resistance plasmid as an alternative and thus applying the antibiotic ClonNAT (150 µg/mL) in the respective steps.

Screening of Diatom Transformants

Schematic outline of the co-transformation of diatoms and selection steps.

Following the transformation, a selection and screening process was performed to identify a successful transformation. Since eGFP is linked to the proteins of interest, fluorescence signal indicates a successful plasmid incorporation. Colonies were selected and picked from the Nourseothricin-containing agar plates. These colonies were subsequently cultivated in a 24 well plate with 2 mL ASW (100 µg/mL Nourseothricin) under previous conditions. To verify the presence of the desired plasmid, each cultivated colony was screened using fluorescence microscopy after four to six days. To enhance the efficiency of positive plasmid identification, colonies showing the desired fluorescence signal were further propagated. This iterative cultivation process (displayed in figure above) was continued until a high degree of positive cells, ideally within the range of 80-100%, was achieved.

In-Gel plating

Cultures with a high eGFP fluorescent signal to non-fluorescent cells were carefully selected and subsequently plated in an in-gel system. To create the in-gel medium, we used 1% clean diatom agar (previously washed with Isopropanol). To prepare the in-gel medium, 2 µL of the desired culture were transferred to a new sterile 15 mL falcon tube. In a separate 50 mL falcon tube, 35 mL of artificial seawater (ASW) were combined. Biotin, KH₂PO₄, and Nourseothricin (at a concentration of 100 µg/mL) to attain the correct concentration for a 50 mL ASW agar stock. Next, 15 mL of preheated 1% diatom agar (at a medium temperature of approximately 70 °C) was transferred to the ASW medium and thoroughly mixed by inversion. A portion of the resulting 0.3% ASW diatom agar mixture (15 mL) was transferred into the 15 mL falcon tube (at a medium temperature of approximately 32 °C). It is crucial to maintain the temperature of the ASW medium at approximately 30 °C to prevent excessively high temperatures, which can be detrimental to diatom cells. The contents of the 15 mL falcon tube were also well-mixed by inversion and then plated onto a sterile petri dish. The petri dish was securely sealed with parafilm and carefully moved to the constant light incubation room, although caution was exercised due to the liquidity of the 0.3% agar. The incubation was continued for 4-6 days, allowing small diatom colonies to emerge. Subsequently, 48 colonies were picked from each petri dish using a 10 µL tip and transferred into a well plate containing 1 mL ASW medium supplemented with Nourseothricin (at a concentration of 100 µg/mL). Further incubation was conducted in a day/night cycle light incubation room. To confirm the presence of the desired plasmid, each cultivated colony was screened using fluorescence microscopy after four to six days.

Influence of Detergents on FAST-PETaseactivity

Prior to the application of a specific detergent in the cell wall isolation process, a detergent evaluation was performed in the context of purified FAST-PETase from heterologous expression in E. coli. This evaluation aimed to identify the most suitable detergent for subsequent use in cell wall isolation. The detergents that were tested included 0.5% Tween-20, 1% Triton X 100, 1% IGEPAL, and 1% SDS. Each detergent was incubated with 1.6 ng/µL FAST-PETase in buffer (50 mM Tris-HCl pH 8.0 and 300 mM NaCl) for one hour before absorption measurements. The SDS sample was incubated at 50°C, the other detergents at 4°C. Following the detergent exposure, enzyme activity was assessed using a colorimetric assay with p-nitrophenyl acetate as the substrate. The measurements were taken at an absorbance of 405 nm, enabling the quantification of enzyme activity by product formation. After evaluation, it was observed that the detergent treatment with Tween-20 had the least impact on enzyme activity, followed by IGEPAL, thus making them suitable choices for the subsequent steps in the cell wall isolation process.

Test cell wall isolation of Thalassiosira pseudonana transformants with Tween-20

The isolation procedure was initially performed using cultures with a minimal proportion of transformants. This approach served the purpose of acclimation to the technique and optimizing conditions before its application to cultures of greater relevance for subsequent activity tests. The entire process was carried out on ice or in a cold room. PMSF, a protease inhibitor, was consistently added to each buffer at various stages.

To prepare the cell suspension, two portions, each containing 2.5 g of T.p. FAST-PETase pellets were placed in separate 50 mL falcon tubes. These pellets were resuspended in 25 mL of freshly prepared Buffer (consisting of 50 mM TRIS-HCl pH 8.0 and 300 mM NaCl). In each falcon tube, 1 mM PMSF was introduced.

The sonication process was performed using a sonication rosette device to maintain low temperatures. Beforehand, the sonicator was cleaned at the conclusion of the sonication process, first with deionized water and then with 80% ethanol. 10 mL of the cell suspension was sonicated, employing a thin sonication (M72). The parameters were set at 20% amplitude, 2 minutes duration, and 30 seconds pulse on/off. The sonication process was manually monitored under a microscope and repeated if necessary or with an increased amplitude. The sonication process comprised 2 rounds at 20% amplitude, 2 minutes each, with 30 seconds of pulse on/off, followed by 4 rounds at 30% amplitude, 2 minutes each, with the same pulse cycle. Finally, 2 rounds at 40% amplitude, 2 minutes each, with the same pulse cycle were executed.

Following sonication, the cell wall suspension was centrifuged in two 50 mL tubes at 3200 x g for 5 minutes at 4°C, and the supernatant was discarded. The cell pellet was washed twice, each time with 25 mL of cold buffer plus appropriate PMFS. Subsequently, a mixture consisting of a 0.5% Tween-20 solution dissolved in buffer was introduced into each falcon tube. These tubes were placed in a rotating wheel (settings: F1; 30 rpm) within a cold room for 1 hour.

After the detergent treatment, the samples were centrifuged at 3200 x g for 5 minutes at 4°C, and the supernatant was discarded. The cell pellets underwent four washing steps, each with 10 mL of buffer and 1mM PMSF. A fifth wash was performed with 20 mL of the same buffer and 1mM PMFS. Following the final wash, 10 mL of buffer and 1mM PMFS was added to each sample, and 1 mL aliquots were prepared. These aliquots were flash-frozen using liquid nitrogen and stored at -80 degrees.

The results indicated an insufficient outcome. The detergent Tween-20 proved unsuitable, as the pellet retained its brown color. IGEPAL as detergent was applied in a second cell wall isolation process.

Test cell wall isolation Thalassiosira pseudonana transformants with IGEPAL

Five 1 mL aliquots of T.p. FAST-PETase Tween-20 were combined into a single 15 mL falcon tube. The volume was increased to 14 mL by adding the previously used buffer and the relevant quantity of PMSF. The mixture was centrifuged under equivalent conditions and the supernatant was removed.

To eliminate residual Tween-20, two cleaning steps were executed, each involving 14 mL of buffer and 1mM PMSF, followed by centrifugation. The supernatant was discarded after each step. The resulting pellet was resuspended in 10 mL of buffer and 1mM PMSF. Using the same M72 sonication tip, two cycles of sonication were performed at 30% amplitude for 30 seconds, with manual interruption. Subsequently, a single cycle was executed at 30% amplitude for 10 seconds. The suspension was adjusted to 14 mL with buffer and PMSF and centrifuged. Three consecutive cleaning steps were carried out, mirroring the previous protocol. The pellet was resuspended in 5 mL of 1% IGEPAL solution in buffer, with the appropriate addition of PMSF. The suspension was subjected to a one-hour incubation in a cold room, placed on a rotating wheel with settings at F1 and 30 rpm. Following the incubation, the mixture was centrifuged, and cleaning steps were executed, aligning with the earlier protocol. The pellet was resuspended in 5 mL with the appropriate PMSF, and 1 mL aliquots were prepared. These aliquots were cryopreserved using liquid nitrogen and stored at -80 °C.

A successful outcome was achieved with IGEPAL exhibiting a bright and clean pellet.

Cell wall isolation Thalassiosira pseudonana transformants with IGEPAL

The isolation procedure was initiated with cultures grown in Nourseothricin, specifically targeting for the transformant FAST-PETase and E. coli Laccase. Four separate harvests of 300 mL per culture type were conducted, and the collected material from each harvest was filtered. Subsequently, the contents were combined into a single 50 mL falcon tube. Each harvest yielded an approximate cell count of 1.5·10⁶ cells/mL. After centrifugation at 3,200 g for 5 minutes at room temperature, the supernatant was carefully discarded, and the pellet was resuspended to a volume of 20 mL in the buffer as described above, along with the addition of 1 mM PMSF.

The sonication process started with a preliminary test at 25% amplitude for 30 seconds. Subsequently, the following sonication cycles were performed: 30% amplitude for 2 minutes with a 30-second pulse on/off, with regular microscopic examination to assess progress.

Following sonication, the cell lysates were centrifuged and the supernatant was discarded. Each sample was resuspended in 30 mL of buffer along with appropriate PMSF. Additionally, four washing steps were performed as previously described. After the final washing step, the supernatant was discarded, and each sample was resuspended to 15 mL in a solution containing 1% IGEPAL in buffer and 1 mM PMSF. The samples were placed on a rotating wheel in a cold room for a one-hour incubation with settings at F1 and 30 rpm. After incubation, the detergent was removed by three washing steps with buffer and 1 mM PMSF. The pellet’s brightness was considered insufficient. Therefore, each 50 mL tube pellet was divided into two 15 mL tubes, with a fill volume of 10 mL in buffer and PMSF. Additional sonication cycles were performed with three cycles at 30% amplitude for 2 minutes and a 30-second pulse on/off for each 15 mL tube. The success was controlled via microscopy. After sonication, three washing steps were executed with a 14 mL volume. This was followed by another incubation in the cold room for one hour with 1% IGEPAL in buffer and PMSF. Five additional washing steps were conducted with a 14 mL volume, resulting in a bright and sufficiently isolated cell wall. The contents of two 15 mL falcon tubes were merged again for each culture type, and the samples were resuspended to a volume of 4 mL. Finally, 1 mL aliquots were prepared and stored at -80°C.

Continuous Activity Test of immobilized FAST-PETase after cell wall isolation

A continuous activity test was carried out to assess the activity of immobilized FAST-PETase using different volumes of cell wall suspension: 200 µL, 50 µL, 25 µL, 5 µL, and 2.5 µL.

The test was conducted in a buffer comprising 50 mM Tris and 300 mM NaCl, along with the addition of 1 mM PMSF and 1 mM p-nitrophenyl acetate as the substrate. Each well was filled with a total volume of 350 µL for the test. The measurement was initiated by introducing the substrate, and absorbance was immediately measured at 405 nm. Measurements were taken over a span of approximately 2 minutes, with intervals of 10 seconds between measurements. Throughout the measuring process, a blank control containing only the buffer and substrate was included to serve as a reference. The presence of disruptive factors in the cell wall suspension impeded the reliability of the continuous test. Consequently, a discontinuous test was planned as an alternative approach serving the purpose to draw definitive conclusions about the enzyme’s activity.

Discontinuous Activity Test of immobilized FAST-PETase and E. coli Laccase after cell wall isolation

A discontinuous activity test was performed to evaluate the performance of immobilized FAST-PETase and E. coli Laccase at diatom biosilica, following the cell wall isolation.

The test was conducted in a discontinuous format, employing 1 mM p-nitrophenyl acetate as the substrate, 1 mM PMSF, and a buffer solution with a composition of 50 mM Tris-HCl pH 8.0 and 300 mM NaCl for the FAST-PETase activity measurement. For the E. coli Laccase, sodium acetate buffer (pH 4, 100 mM) and 1 mM ABTS as well as 1 mM PMSF were applied. Different volumes of the cell wall suspension, carrying the immobilized FAST-PETase and E. coli Laccase, were used: 300 µL, 200 µL, 50 µL, 25 µL, 10 µL, and 2.5 µL. The samples were incubated at 24°C for a total duration of 90 minutes, with constant shaking at 650 rpm in a thermocycler. Measurements were taken after 0 min, 15 min, 30 min, 60 min and 90 min. Each sample was prepared with a total volume of 400 µL in a 2 mL tube. The buffer volume was adjusted relative to the volume of the applied cell wall suspension. Before each measurement, the sample was centrifuged for 1 minute at approximately 20,000 x g. Subsequently, 350 µL of the supernatant was transferred into a 96-well plate for analysis. After each measurement, the respective supernatant was put back into its equivalent tube and brought back into suspension through vortexing. Throughout the entire measuring process, a blank control was included, consisting of the buffer and the substrate p-nitrophenyl acetate or ABTS. The hydrolysis of p-nitrophenyl acetate was controlled via absorption measurement at 405 nm, the oxidation of ABTS at 420 nm.

During the FAST-PETase test, it was observed that the FAST-PETase blank displayed a significant autohydrolysis effect, which affected the accuracy of activity measurements. Consequently, the discontinuous test was repeated under the same conditions, with one adjustment - the temperature was increased to 50°C, which aligns with the optimal temperature for FAST-PETase (Lu et al. 2022). All tests were performed in a single determination due to the limited amount of cell wall suspension available.

Discontinuous Activity and Inhibition Test of immobilized E. coli Laccase

Subsequent to the previous tests, a similar activity test was conducted for immobilized E. coli Laccase. In this instance, adjustments were made, including a test volume of 100 µL for the cell wall suspension. Triple determinations were performed for both the blank and the laccase. In addition, to confirm the origin of the oxidized ABTS observed in the prior discontinuous test, a laccase inhibitor, sodium azide, was introduced to the same quantity of immobilized laccase, with a concentration of 1 mM (Johannes and Majcherczyk 2000). This served to ascertain that the observed oxidation of ABTS was indeed a result of laccase activity and not attributable to any other substances attached to the diatom's biosilica. Measurements were taken at 0 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, and 90 minutes.

PET Degradation Test

To facilitate contact between the enzyme and the PET substrate for degradation analysis, microplastic was generated from PET film. The PET film was cut into pieces, and a partial breakdown was achieved by adding liquid nitrogen and using a mortar. Subsequently, the microplastic underwent a thorough cleansing process involving washing with 1% SDS, followed by 20% ethanol, and finally, deionized water. The microplastic was then placed in a thermos cycler at 60°C until complete drying. Afterwards, it was reconstituted in a buffer composed of 50 mM Tris-HCl pH 8.0, 300 mM NaCl to ensure even distribution.

100 µL cell wall suspension carrying the FAST-PETase enzyme was introduced into a suspension containing approximately 1.4 mg of the prepared microplastic. The volume was adjusted to 500 µL with a buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl) and 1 mM PMSF. Samples were initially collected after the mixing step and then at intervals of 4 hours, 24 hours, and 48 hours. A triple determination of each sample was performed. To terminate the enzyme reaction, the samples were subjected to heat treatment at 85°C for 20 minutes. Any remaining components were removed through centrifugation (20 min at room temperature at 10,000 x g). Prior to HPLC analysis, the samples were filtered through a 0.2 µm nylon syringe filter.

Diclofenac Degradation Test

Investigating the laccase enzyme activity by degrading diclofenac, 100 µL cell wall suspension carrying the laccase enzyme was combined with diclofenac (500 µM final concentration) in sodium acetate buffer (100 mM, pH 4) and 1 mM PMSF. Sampling was initiated immediately after the mixture of these components, followed by additional sampling at time intervals of 4 hours, 24 hours, and 48 hours. To stop the enzyme-catalyzed reaction, the samples were subjected to heat treatment at 85°C for a duration of 20 minutes. Following this, the samples were centrifuged at room temperature with a force of 10,000 x g for a duration of 20 minutes. In preparation for HPLC analysis, each sample underwent filtration using a 0.2 µm nylon syringe filter.

Microscopy

2 ml of T. pseudonana culture with appr. 1*10⁶ were concentrated by centrifugation at 4000 rcf. 2 µl of the concentrated cell suspension were placed on a cover slip. The droplet was fixed using a appr. 5 mm thick slice of a 1 % DNA grade agarose gel in ASW. Confocal images were obtained by a Zeiss LSM 980 Airyscan 2 (Carl Zeiss AG, Jena, Germany) using the C-Apochromat 40x/1.2 W Corr M27 objective (water immersion). eGFP was excited at 488 nm and detected at 420-480 nm with the Airyscan detector. Chlorophyll A auto-fluorescence was excited at 655 nm and detected at 607-720 nm with the Airyscan detector. Brightfield images were detected with the T-PMT detector at 300-900 nm.

Growth curves

Cell density of pre-cultures of T. pseudonana was counted using a Bio-Rad TC20 Automated Cell Counter (Bio-Rad Laboratories, Inc., Hercules, USA). Triplicates were setup by inoculation of 100 ml ASW with 1*10⁶ cells each. Cell were counted using the TC20 Automated Cell Counter. OD600 measurements were blanked against de-ionized water after confirmation that there is no difference in absorption at 600 nm between ASW and de-ionized water.

20 l cultivation

Two 20 l tanks each were inoculated with 40 ml of a pre-culture of T. pseudonana tpSil3SP56-eGFP-FAST-PETase (1.87 * 10⁶ cells/ml) and a pre-culture of T. pseudonana tpSil3SP56-eGFP-E. coli Laccase (1.01 * 10⁶ cells/ml). Cells were harvested after 8 days using continous flow centrifugation, pellets frozen with liquid nitrogen and stored in -20°C until further use.

Image analysis

Maximum projections and reslicing of confocal image stacks were performed using Fiji.