Part:BBa_K4750003

SP+T8 (tpSil3)_eGFP_FAST-PETase

This part is composed of a fragment of the coding sequence of SP+T8 (BBa_K4750001), the coding sequence of eGFP (BBa_K1123017) and the coding sequence of FAST-PETase (BBa_K4750002).

Target organism: Thalassiosira pseudonana
Main purpose of use: in vivo immobilization of FAST-PETase using the method LiDSI (Poulsen et al. 2007) for the degradation of microplastic.

Usage

This composite part can be used for expression in Thalassiosira pseudonana. Due to the N-terminal SP+T8 sequence, the fusion protein becomes entrapped in vivo in the biosilica cell wall of T. pseudonana. The underlying method was coined Live Diatom Silica Immobilization (LiDSI) (Poulsen et al. 2007). The eGFP sequence serves as a linker and reporter to confirm immobilization in the cell wall using fluorescence microscopy. The immobilized FAST-PETase is designed to be used in bioremediation to degrade microplastic PET either using the living diatom or the isolated biocatalytically active cell wall. Studies indicate that LiDSI-immobilized enzymes (e.g. Glucose oxidase) do not get released from the biosilica and profit from an increased stability (Sheppard et al. 2012). This has yet to be shown, for this part.

Biology

The N-terminal tpSil3 fragment (BBa_K4750000) consists of a 17 amino acid long N-terminal signal peptide (SP) and a 37 amino acid long sequence termed T8 (Sheppard et al. 2012). The SP is responsible for cotranslational import into the endoplasmic reticulum (Sheppard et al. 2012). The intracellular route of the fusion protein appears to involve the endoplasmic reticulum as well as the Golgi apparatus (Poulsen et al. 2013), before being incorporated into the biosilica within the silica deposition vesicle. Poulsen et al. showed that tpSil3_eGFP fusion proteins become immobilized in the valve and girdle bands. Their results suggest that the fusion protein is protected by the biosilica, but not completely enclosed. (Poulsen et al. 2007). The Functional, Active, Stable and Tolerant PETase (FAST)-PETase is a variant of the PETase isolated from Ideonella sakeiensis (Yoshida et al. 2016) bearing the mutations: N233K, R224Q and S121E. It was engineered using a self-supervised, 3d convolutional neural network (Lu et al. 2022). The mutations resulted in increased thermal stability and overall degradation of PET. PETases are hydrolases acting on poly(ethylene terephthalate) (EC 3.1.1.101). The reaction primarily releases mono(2-hydroxyethyl) terephthalic acid (MHET) with terephthalic acid (TPA) and bis(2-hydroxyethyl) TPA (BHET) as side products (Yoshida et al. 2016).

Characterization of active biosilica

The construct was assembled using Gibson Assembly, cloned into the pTpNR T8 vector and replicated in E. coli DH5α. Tungsten particles were coated with the isolated plasmid. Thalassiosira pseudonana CCMP1335 was transformed using the Particle Delivery System PDS-1000/He (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The transformation was performed as a co-transformation with pTpFCP/NAT, which harbors the nourseothricin resistance gene. Fluorescing colonies were picked and subject to at least one screening cycle (colony-picking, liquid culture, fluorescence screening, in-gel plating). Detailed information can be found on https://2023.igem.wiki/tu-dresden/experiments.

Biosilica isolation

Cell wall isolation Thalassiosira pseudonana transformants with IGEPAL
The isolation procedure was initiated with cultures grown in Nourseothricin (ClonNAT), specifically targeting for the transformant. Four separate harvests of 300 mL Thalassiosira pseudonana transformat were conducted, and the collected material from harvest was filtered. Subsequently, the contents were combined into a single 50 mL falcon tube. Each harvest yielded an approximate cell count of 1.5·10e6 cells/mL. After centrifugation at 3200 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 (50 mM TRIS, 300 mM NaCl, pH 8), along with the addition of PMSF (1 mM total) as protease inhibitor. 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 as described above along with appropriate PMSF. Additionally, four washing steps were performed, each with a volume of 20 mL buffer with approritate PMSF. 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 (4°C) 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 as described above. 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 (for further information see (BBa_K4750007)) 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, and the samples were resuspended to a volume of 4 mL. Finally, 1 mL aliquots were prepared and stored at -80°C.

Activity Assays

The activity of PETases can be assessed with several methods as reviewed by Pirillo et al. Among the photometric assays, colorimetric determination of the hydrolysis of p-nitrophenol esters are most commonly used. To analyze the hydrolysis of PET by PETase, reverse-phase high performance liquid chromatography (HPLC) can be used (Pirillo, Pollegioni, and Molla 2021). To assess the activity of the immobilized FAST-PETase we used a colorimetric assay using p-nitrophenyl acetate as a substrate and C18 reverse-phase HPLC to assess the degradation of microplastic.

Continuous Photometric assay using p-nitrophenyl acetate

PETase activity can be measured by the hydrolysis of p-nitrophenyl acetate yielding p-nitrophenol (pNP) and acetate. In alkaline conditions (pH > 7.15 [pKa of pNP]), pNP becomes deprotonated. The phenolate leads to an increase in absorbance at 405 nm (molar extinction coefficient: ε405 = 18,400 nm−1·M−1). An initial continuous activity test was performed at first to evaluate the functionality of immobilized Fast-PETase. This test involved varying volumes of cell wall suspension, namely 200 µL, 50 µL, 25 µL, 5 µL, and 2.5 µL. The experiment started by introducing the substrate, followed by immediate absorbance measurements. However, the presence of disruptive factors within the cell wall suspension posed significant challenges, rendering the results of the continuous test inconclusive, as no enzyme activity was detectable during the entire measurement period. Instead, what became evident was a pronounced autohydrolysis of pNPA within the buffer.

Fig. 1, 2 pNPA assay with clean cell wall immobilised FAST-PETase at 50°C

Discontinuous Photometric assay using p-nitrophenyl acetate

In response to the results of the continuous activity test, a discontinuous test was devised as an alternative strategy to facilitate the attainment of definitive conclusions regarding the enzyme's activity. Various volumes of the cell wall suspension, each containing immobilized FAST-PETase (ranging from 2.5 µL to 300 µL), were systematically examined to identify the most suitable volumes for subsequent degradation experiments, with the goal of optimizing enzyme activity over time. Throughout the entire measurement process, a blank control, comprising the buffer and the respective substrate (p-nitrophenyl acetate), was consistently integrated. Notably, during the FAST-PETase assessment, a substantial autohydrolysis effect was observed in the FAST-PETase blank of pNPA, significantly impacting the accuracy of activity measurements. Consequently, the discontinuous test was repeated under identical conditions but increased temperature (50°C known to be the optimal temperature for FAST-PETase activity)(Lu et al. 2022). Under both 24°C and 50°C conditions, no definitive conclusion regarding the existence or non-existence of Fast-PETase activity could be drawn due to the pronounced autohydrolysis of the pNPA substrate.

Fig. 3 pNPA assay with suspension of silica immobilised enzymes using different volumes

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.

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Fig. 4 Growth determination of T. pseudonana strains using different methods

Fig. 5 light microscopy of eGFP fluorescent and non fluorescent T. pseudonana cells

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 172
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 143
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1116
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 815