Wet Lab

Strain Selection

There are several species of bacteria capable of degrading microplastics, mainly those that form a biofilm on the surface of polyethylene [1]. The two of the genera that have shown more promising results in the biological degradation of both PET and PE are the Bacillus and Pseudomonas. Pseudomonas spp. alone were reported to degrade 35 different plastic types, including most of the mass-produced polymers [2,3] with the species Pseudomonas aeruginosa being one of the most efficient PET degradation bacteria [1]. However, in this project, the Bacillus subtilis species was chosen, which by itself has a low PET degradation activity but, due its ease of transformation it allows the expression of the efficient PETase and MHETase enzymes of Ideonella sakaiensis. Moreover, the B. subtilis secretion system is well-known and was already studied and industrially implemented to secrete a wide variety of enzymes. Finally, its ability to use PE as a carbon source for its growth will lower the culture maintenance costs.

Materials and Methods

Cloning Procedure

Codon-optimized genes for expression in Bacillus subtilis were obtained from IDT (Iowa, USA). The genes used in this study were amplified by polymerase chain reaction (PCR) using Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Waltham, USA) in a LifeECO Thermal Cycler (Bioer Technology, Zhejiang, China). All primers were purchased from IDT. DNA fragments were purified using DNA Clean and Concentrator DNA Kit (Zymo Research, Irvine, USA). Plasmids were extracted using Plasmid Miniprep kit (Zymo Research). Clones were obtained by performing FastCloning [4] and transformed by heat-shock in chemically competent E. coli NEB 5-alpha cells (New England BioLabs, Massachusetts, USA). The success of ligation was visually checked by picking white colonies instead of pink ones – due to the substitution of the gene RFP in positive clones – and further confirmed by sequencing (StabVida, Lisbon, Portugal). Protocols were performed in accordance with manufacturer’s instructions.

For long-term storage, glycerol was added to a final concentration of 30 % (v/v) to overnight cultures in selective media and kept in a -80 °C freezer.

Plasmid Construction

Our plasmid constructs use the pBS1C and pBS2E plasmids as vectors to express our target gene sequences. We used two different chassis, each containing one of our insert sequences, featuring either PETase or MHETase sequences, so that we could select the cell strains that contained both PETase and MHETase, using the two different antibiotic resistances conferred by these plasmids. Both of our insert sequences feature a similar design, only changing the target protein sequence.

Analysing in detail our insert sequences, they start with the P43 promoter sequence - a strong constitutive promoter already validated for gene expression in Bacillus systems which activates the transcription during exponential and lag phases of growth. Followed by the ribosome binding site (RBS), this sequence in bacterial mRNA ensures the translation of proteins in Bacillus subtilis. Then, the native signal peptide of each enzyme, present in our design, ensures that both enzymes, PETase and MHETase, are properly secreted to the extracellular matrix. In the case of PETase an additional signal peptide was tested, the SPamy. Located between the signal peptide sequence and the T7 terminator is the target protein sequence which will be further detailed below for both our enzymes. Our insert sequence design is then finalized by a rho-independent T7 terminator (T7Te) which encodes for an RNA sequence that can form a stable stem-loop structure followed by a run of six uridylate residues thus allowing for an efficient transcription termination.

PETase is a hydrolase, EC 3.1.1.101, with a polyethylene terephthalate degradation capability. This enzyme was first discovered in the plastic degrading bacterium Ideonella sakaiensis as a part of a two-enzyme system, responsible for the hydrolysis of the polymer into a heterogeneous mixture of the respective constituents [5]. Additionally, a PETase codon-optimized sequence for B. subtilis , the W159H/F229Y mutant displays an optimal compromise between the catalysis (K_cat) and the Michaelis-Menten (K_M) constant, being 9.64 s^-1 and 0.08 mmol/L, respectively. The development of the optimized enzyme was made possible by the findings of Xiangxi’s Group [6]. This mutant together with the signal peptide, allows for the excretion of PETase by B. subtilis and a fast and efficient PET degradation inside the bioreactor.

MHETase is a hydrolase, EC 3.1.1.102, with a mono-(2-hydroxyethyl) terephthalic acid degradation capability. It is the second constituent of the two-enzyme system used by the plastic degrading bacterium I. sakaiensis to degrade PET into its constituents terephthalic acid (TPA) and ethylene glycol (EG). Additionally, a MHEtase sequence codon-optimized for a B. subtilis chassis with a W397A mutation displays an optimal compromise between the catalysis (K_cat) and the Michaelis-Menten (K_m) constant, being 15.35 s^-1 and 1.0 μmol/L, respectively. This mutant was developed by Gottfried’s Group [7].

To further improve our system we aimed to design constructs containing the improved mutants, not just the wildtype enzymes, for both enzymes, however that was not possible during the course of our project. Our constructs are presented in Figure 1 and detailed in Table 1.

The primers used to amplify the insert sequences in the FastCloning protocol have homologous regions with the chassis used that were automatically calculated using the NEBuilder software and are presented in Table 2. This FastCloning protocol uses the specifically calculated primers in two separate PCR reactions, one for the vector and another for the insert. It is believed that the exonuclease activity of the DNA polymerase produces sticky ends in both PCR products, hence allowing us to fully assemble the construct just by mixing the two PCR products. Finally, this mixture is incubated with a methylation sensitive restriction enzyme to remove the templates and transformed into competent E. coli cells.

Bacillus subtilis strains

Bacillus subtilis W168 and Bacillus subtilis KO7 were used as hosts for gene expression. B. subtilis belongs to the genus Bacillus and being a member of Gram-positive bacteria, the lack of outer membrane enables the direct secretion of proteins to the growth medium. These proteins are completely transported across the B. subtilis membrane as long as they contain an amino-terminal signal peptide that enables them to be secreted. As such this ease of transportation of proteins makes B. subtilis of interest in several areas of biotechnology [7].

In this project two strains of B. subtilis W168 and B. subtilis KO7, were used as hosts for gene expression and an E. coli strain was used as an intermediate host. Characteristics of these strains, as well as of the strains produced by the transformation of the latter with our constructs, are presented in Table 3.

The W168 strain, is characterized by being auxotroph only to tryptophan making it one of the most commonly used in academic research and industrial processes due to its ease of transformation providing the basis for B. subtilis chassis characterisation [8,9].

The KO7 strain was chosen to be the chassis for the project. This strain’s genotype is characterized by having deletions in 7 protease genes found in WT B. subtilis (ΔnprE, ΔaprE, Δepr, Δmpr, ΔnprB, Δvpr and Δbpr) that code for 7 different extracellular proteases responsible for protein degradation [10]. This leads to a higher half-life of the extracellular protein of interest in this project.

The single plasmid transformations were performed following the protocol developed by the Uppsala team last year, presented in the Protocols section.

Concomitantly with the results obtained by Uppsala team, hundreds of colonies were obtained in the plates corresponding to the pBS1C transformation, but only a single colony was obtained when using pBS2E (using B. subtilis KO7 as host strain). No colonies were obtained when transforming the two plasmids simultaneously. So, the approach suggested but not tested by the Uppsala team was implemented: transforming the cells with pBS2E first and only then with pBS1C. Moreover, small modifications to the Uppsala protocol were employed by culturing the pre-inoculum in LB medium instead of SP medium and excluding the plasmid linearization step. Hundreds of double mutant clones were obtained by implementing this two-step protocol.

Growth Experiments

A single colony was picked from LB plates and inoculated in 10 mL of liquid LB medium with the appropriate antibiotic(s). The pre-cultures were grown aerobically on a rotary shaker at 37 ºC and 180 rpm, overnight. An appropriate volume of cells was harvested from the pre-culture by centrifugation (10 min at 3000×ɡ), resuspended and then transferred to 250 mL shake flasks with 50 mL of LB medium supplemented with the appropriate antibiotic(s) concentration, yielding an initial optical density at λ600 nm (OD600) of 0.1. These cultures were also cultivated on a rotary shaker at 150 rpm at 28 ºC for 24 h. All cell optical density measurements at 600 nm were performed using the spectrophotometer Ultrospec 10 from Biochrom (Cambridge, UK).

SDS-PAGE Analysis

After growing for 24 h in shake flasks, a medium sample of each strain was collected and the optical density at λ600 nm (OD600) was measured. For each strain, 1 mL sample with a final OD600 of 0.6 was used for the following steps.

Intracellular Protein Fraction

Cells were harvested in a microcentrifuge at 5000xg, 10 min. Supernatants were transferred to new tubes to be later used for the extracellular protein extraction protocol, leaving cell pellets intact. Pellets were frozen at -20 ºC for 30 min and then digested with 25 μL digestion buffer, composed of NZY Bacterial Cell Lysis Buffer (NZYTech, Portugal), lysozyme and DNase I (2 µL of lysozyme at 50 mg/mL and 2 µL of DNase I per 1 mL of NZY Bacterial Cell Lysis). Samples were shaken at 200 rpm for 20 min at room temperature. 8.3 μL (in a 1:4 ratio) of Laemmli Sample Buffer (composed of 4x Laemmli buffer and 10 % [v/v] β-Mercaptoethanol) was added to each sample, the resulting mixture was then incubated at 98 ºC for 5 min.

Extracellular Protein Fraction

10 % (v/v) of trichloroacetic acid (TCA) was added to each supernatant and proteins were allowed to precipitate on ice for 30 min. Samples were centrifuged at 14000 rpm, 5min and supernatant was discarded. Precipitated protein pellets were washed with cold acetone: 200 μL of acetone was added to each tube and tubes were centrifuged again at 14000 rpm, 5 min and acetone was discarded; this step was performed twice. Tubes were incubated at 90 ºC, 5 min, open and inside the hood, to evaporate leftover acetone and dry off pellets. Protein pellet was solubilized in 25 μL of 1X SDS-Glycine Buffer. 8.3 μL (in a 1:4 ratio) Laemmli Sample Buffer (composed of 4x Laemmli buffer and 10% [v/v] β-Mercaptoethanol) was added to each sample, which was then incubated at 98 ºC for 5 min.

SDS-PAGE Gels

10% SDS-PAGE gels were prepared and 10 μL of each sample and 3 μL of NZYColour Protein Marker II (NZYTech, Portugal) were loaded into the SDS-PAGE gels. Gels were run at 200 V for approximately 1 hour, using 1X SDS-Glycine Buffer. Gels were colored with BlueSafe (NZYTech, Portugal).

PETase and MHETase have a molecular weight of 27.3 kDa and 63.1 kDa, respectively.

BHET degradation assay

The experiment to evaluate potential of PET hydrolysis by the developed strains was adapted from [11] using the analogue BHET as substrate. The Bacillus strains used in this study (Table 3) were grown in LB medium at 28 ºC in shake flasks as described before, after 24 h a medium sample of each strain was centrifuged for 15 min, 4 ºC at 15000 rpm to collect the supernatant to be used as the crude enzyme solution. A reaction mixture of 600 μL was prepared with the following final concentrations: 0.9 mM BHET; 40 mM Na₂HPO₄·HCl pH7; 80 mM NaCl; 20 % (v/v) DMSO and 2 % (v/v) crude enzyme solution. In a second experiment, the crude enzyme solution was increased to 50 % (v/v). This mixture was incubated at 85 ºC for 2 h, the reaction was stopped by adding 9.6 μL of Na₂HPO₄·HCl (pH 2.5) and 120 μL of DMSO, followed by an incubation step of 10 min at 85 ºC. The samples were then filtered (2.2-μm filter) to be further analyzed by HPLC.

HPLC Method

The High Performance Liquid Chromatography (HPLC) method was adapted from [12]. The compounds were quantified using HPLC from Waters (Massachusetts, USA) model ALLIANCE 2695 equipped with an ultraviolet detector model Waters 486 and a symmetry C18 reverse phase column (5 µm 4.6 x 250 mm) of pore size 100 Å. For the analysis, 5 μl of sample was injected using a gradient solution with three solvents: (A) ultra-pure water; (B) Methanol and (C) ultra-pure water with glacial acetic acid 1 % (v/v). The fraction of B decreased linearly from 60 % until 55 % in 10 % of Buffer C from 15 min after injection, returning to the initial conditions for 8 min to equilibrate the column. The flow rate was 1 mL/min. The column temperature was maintained at 25 ºC. BHET and TPA were analyzed at 240 nm.

The calibration curves of TPA and BHET were obtained by injecting standards with known concentrations for each metabolite. Metabolite concentrations in samples were calculated by comparing the peak areas of the samples with the calibration curves.

Results

Growth Curve

In Figure 2, the growth-curve for the different strains developed in this work are depicted. These curves were obtained by measuring the optical density of the cultures at different time points.

Analysing the growth-curves for the different strains depicted in Figure 2, it is possible to see that the final maximum concentrations achieved in the stationary phase are not very different between each strain. The growth profile of the different strains seem similar, with the exception of BS1 and BS2 strains. Particularly, the lag phase was prolonged when compared with the wild-type KO7. So, it seems that the expression of PETase with both signal peptides had delayed the initial growth. Strangely, this behaviour is not observed when expressing MHETase as well (strain BS6).

SDS Gel

In Figures 3 and 4 are presented the SDS-PAGE gel results of the different developed strains. Both intracellular and extracellular fractions were analyzed.

After analyzing the Figures 3 and 4, we conclude that in the extracellular fraction of the W168-based strains (wells B and C), a band of the expected size of PETase is visible at 24 h for both signal peptides tested, which is not present in the wild-type strain (well A). Regarding the KO7 cells, no conclusive results were obtained since bands of the expected size are visible in both wild-type and mutant strains for PETase and MHETase.

Calibration Curves

The calibration curves obtained had a R² superior to 0.99 validating the HPLC method implemented to quantify BHET and TPA.

BHET degradation experiment

Since the initial concentration of BHET supplemented to the reaction mixture was 0.9 mM and the presence of TPA was not detected in all the analyzed samples, we conclude that BHET was not hydrolyzed by the enzymes secreted by the engineered strains in this experiment. Analyzing the results presented in Table 4, we can see that all BHET concentration values are very similar among all strains (including wild-type) and comparable to the initial concentration despite the strain or the final concentration of crude enzyme added to the reaction mixture.

We hypothesize that the amount of enzyme available in the supernatant was not sufficient to degrade BHET and to yield TPA, since bands with the expected protein sizes are barely visible in the SDS-PAGE gels. Even in the experiments where the amount of crude enzyme supernatant was increased to 50 % (v/v) – Experiment B – instead of 2 % (v/v) – Experiment A – no variation in the BHET concentration was obtained. Although it is not referred to in the literature, probably an enzyme concentration step is required in these experiments. We did not find a suitable protocol to apply since all the concentration methods usually imply the enzyme precipitation (like the one described in the preparation of the SDS-PAGE gels) turning unfeasible to further catalytic experiments.

General Conclusions & Future Perspectives

Although the developed strains failed to degrade BHET, we believe that the optimal conditions to obtain a sufficient amount of enzyme were not achieved due to the lack of time. The conditions used in this work were adapted from the literature where other strains, plasmids and molecular biology designs were implemented. So, the immediate step should be to test various culture conditions and measure the enzyme concentration for several time points to identify the optimal culture conditions for excreting PETase and MHETase by the different strains. We also envision that the engineered cells could be incubated with PET films for several days and further analyze the surface of the films by Scanning electron microscope (SEM) to evaluate the effectiveness of the secreted enzymes in more realistic conditions.

Protocols

Competence Development & Transformation in Bacillus subtilis (Team UofUppsala 2020)

Day 0:

From a single colony, inoculate 5 mL of SP medium in a tube and incubate at 37ºC O/N with aeration and 150rpm

Day 1:

Dilute 1:50 of the O/N cultures on a 16×125 mm test tube (or equivalent) with 500 µl of fresh SP medium
Add 1ug of DNA and incubate at 37ºC with aeration, 150 rpm, 5h30min
Centrifuge the culture and resuspend in 1 mL of LB medium (pre-heated at 37ºC). Centrifugation + Resuspension is done in an Eppendorf, but the incubation is done in the same tubes as the previous steps.
Incubate for 1h30min at 37ºC with aeration and 150 rpm;
Plate the whole culture in desired media. Resuspension in 50µl recommended SP.

Medium Preparation

SP Medium (for 10 mL)

Spizizen salts 9.215 mL
Glucose 50% (w/v) 400 μL
Casein hydrolysate 5% (w/v) 200 μL
Tryptophan 5mg/ml 100 μL
Ammonium ferric citrate 22mg/mL 5 μL
Potassium glutamate 40% (w/v) 50 μL
MgSO₄ 1M 30 μL

Spizizen Salts (for 1 L)

K₂HPO₄ 14 g
KH₂PO₄ 6 g
Trisodium citrate 1 g
ddH₂O to 1 L

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