Part:BBa_K5117020
BsRBS-BhrPET
This part serves as translational unit composed of the ribosome binding site of Bacillus subtilis (BBa_K5117000) and the gene of the uncultured bacterium HR29 (BBa_K51170010) encoding a polyethylene terephthalate hydrolase (PETase, EC 3.1.1.101), codon optimized for Bacillus subtilis (Xi et al. 2021) and including a secretory signal peptide SPaprE.
Biosafety level: S1
Target organism: Bacillus subtilis
Main purpose of use: Testing enzyme functionality in the host B. subtilis
Potential application: Degradation of PET
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 594
Illegal AgeI site found at 639 - 1000COMPATIBLE WITH RFC[1000]
Enzyme characterization according to literature
The characterization of the enzyme included in this composite part can be found on the basic part page (BBa_K5117010) of the enzyme.
Construct Design
For compatibility with the BioBrick RFC[10] standard, the restriction sitesEcoRI, XbaI, SpeI, PstI and NotI were removed from the coding sequence (CDS). To make the part compatible with the Type IIS standard, BsaI and SapI sites were removed as well. This was achieved by codon exchange using the codon usage table of Bacillus subtilis (Codon Usage Database Kazusa). Upstream from the CDS, there is the ribosome binding site (RBS) for the host Bacillus subtilis followed by a 7 bp spacer. This composite part composed of RBS and CDS was flanked by the BioBrick prefix and suffix sequences and ordered via gene synthesis from IDT.
Construction of a template plasmid
In order to create a template from which this part could be amplified, the part was subcloned into a small vector pSB1C3 (Part:pSB1C3). For that purpose, the plasmid was isolated from E. coli DH10β, yielding a DNA concentration of 431.0 ng/µl. Afterwards, a Backbone PCR with pSB1C3 (Fig. 1) was performed, followed by a restriction digest of the amplified vector backbone and the part with EcoRI and PstI, which were purified via the HiYield® PCR Clean-up/Gel Extraction Kit (SLG, Germany), resulting in DNA concentrations of 32.6 ng/µl (digested pSB1C3) and 14.9 ng/µl.
After ligation, the plasmid was transformed into chemically competent E. coli DH10β cells. Transformants were selected by chloramphenicol resistance (35 µg/ml chloramphenicol) encoded on the pSB1C3 backbone. For the negative control, no DNA was added during the transformation procedure leading to no colony growth on selection plates. For the positive control, cells were transformed with the vector pSB1C3 resulting in a pink bacterial lawn due to the original RFP insert. On the selection plates of the target construct, white colonies were tested for the presence of the correct insert by Colony PCR and agarose gel electrophoresis (Fig. 2).
Colonies with a band at the correct size of the insert were chosen for plasmid isolation according to the HiYield® Plasmid Mini DNA Kit (SLG, Germany). Finally, the plasmid was verified via sequencing by Microsynth Seqlab GmbH (DNA concentration: 112.8 ng/µl).
Cloning into inducible expression vectors
For testing the functionality and activity of this enzyme, the part was cloned into xylose-inducible expression vectors in order to overexpress the gene of interest. Replicative (pBS0E-xylR-PxylA) and integrative (pBS2E-xylR-PxylA) vectors were used, both with a xylose-inducible promoter for induced expression and a xylose repressor to decrease basal promoter activity (Popp et al. 2017). Whereas replicative plasmids provide a high copy number and result in high concentrations of target proteins, genomic integration (in this case into the lacA locus) ensures high stability but results in lower protein concentrations. The vectors were isolated from E. coli DH10β, resulting in DNA concentrations of 112.4 ng/µl pBS0E-xylR-PxylA and 149.9 ng/µl pBS2E-xylR-PxylA. These vectors were digested with EcoRI and PstI and purified using the HiYield® PCR Clean-up/Gel Extraction Kit (SLG, Germany).
The enzyme part was amplified via PCR (Fig. 3) using the plasmid template pSB1C3-BhrPET and subsequently digested and purified via PCR clean up.
After ligation, the plasmids were transformed into chemically competent E. coli DH10β and transformants were selected by ampicillin resistance (100 µg/ml ampicillin) encoded on the vector backbone.
White colonies transformed with the expression plasmids were analyzed by Colony PCR and agarose gel electrophoresis (Fig. 4). Colonies with a band at the correct size of the insert were chosen for plasmid isolation. Finally, the replicative expression plasmid pBS0E-xylR-PxylA-BhrPET and the integrative expression plasmid pBS2E-xylR-PxylA-BhrPET were verified by sequencing and successfully generated (DNA concentrations: 428.2 ng/µl, 124.6 ng/µl).
Ultimately, these expression plasmids were transformed into the target host B. subtilis. Since this part was included in the strategy focused on the secretory expression of target enzymes, WB800N was chosen as an expression strain. This genetically engineered variant of B. subtilis W168 features the disruption of all extracellular proteases. The eight-extracellular-protease-deficient mutant is widely used in industrial applications, as it increases the stability of secreted proteins (Jeong et al. 2018).
The transformants were selected by MLS resistance (1 micro;g/ml erythromycin and 25 micro;g/ml lincomycin) encoded on the vector backbones.
The Bacillus transformation was carried out with early addition of DNA to growing WB800N cells (at OD600 ≈ 0.7) to not miss the timepoint of competence. Afterwards, cells were grown until OD600 ≈ 1.1-1.3 and the same procedure was followed as in the initial protocol (see Experiments page).
The expression plasmids could be transformed into WB800N and colonies were verified by Colony PCR. The presence of replicative plasmids was tested by two primer pairs (double check), whereas both upstream and downstream integration into the lacA locus was checked for integrative plasmids (Fig. 5). Two colonies each with the correct insert size were chosen for cryo-conservation, serving as biological duplicates.
Expression of PETases
For the expression and testing of heterologously expressed enzymes in B. subtilis, we induced protein production in cultures by adding 0.5 % xylose after reaching an OD600 of 0.5 – 0.6. After 24 hours, the supernatants were collected to test secreted PETases. The enzyme activity of the supernatants was tested, with additional samples analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm protein expression. Detailed procedures are available on the Experiments page.
From now on, we will refer to B. subtilis WB800N transformed with the replicative vector pBS0E-xylR-PxylA containing one of our enzymes, such as BhrPET, as "pBS0EX-BhrPET." Similarly, B. subtilis WB800N transformed with the integrative plasmid pBS2E-xylR-PxylA containing the same enzyme will be referred to as "pBS2EX-BhrPET."
Continuous pNPAC assay for determination of activity
To demonstrate the functionality of the produced BhrPET, a continuous p-nitrophenyl-acetate (pNPAc) assay was conducted using supernatants containing this enzyme. In this assay, pNPAc is degraded to acetate and p-nitrophenol (pNP), which imparts a yellow color to the solution (Kademi et al. 2020). Notably, the reaction also occurs in the absence of enzymes, which is referred to as autohydrolysis. To rule out the influence of autohydrolysis on the measured activity values, a negative control without BhrPET was included. Additionally, supernatants obtained from an untransformed B. subtilis WB800N were used as controls to check the ability of B. subtilis to degrade pNPAc natively. The reaction was performed in 50 mM phosphate buffer with pH 7 at 40 °C for 10 minutes (see Experiments page). The results are shown in Fig. 6.
The volume activity was calculated with extinction coefficient ε = 6.52 mM-1cm-1 (Kademi et al. 2020). Notably, the influence of DMSO on the pNP absorbance was neglected. In the future, to obtain more precise values for volume activity, the extinction coefficient should be determined experimentally for the exact conditions used. However, since the focus of our project was on the enzyme immobilization on spore surface of B. subtilis, no further assay optimizations were performed.
With pNPAc assay no background activity could be detected with supernatants obtained from B. subtilis WB800N, while the activity of the BhrPET-containing supernatant was successfully demonstrated, with an activity of 0.375 U/ml. Interestingly, supernatants from the uninduced culture showed slightly higher activity compared to the WB800N controls, indicating basal expression of the vector.
Determination of molecular weight
To determine the molecular weight of the expressed proteins, a 12 % SDS-PAGE was performed for the PETases. Samples were mixed with SDS loading buffer and heated to denature the proteins. The samples and molecular weight marker (PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa) were then loaded, and the gel was run at 100 V for approximately 1.5 hours. Gels were stained with Coomassie Blue and destained to visualize the protein bands. Further details of the procedure can be found on the Experimentspage.
For the analysis of the molecular weight of BhrPET, we used both generated strains (pBS0EX-BhrPET and pBS2EX-BhrPET). The expected molecular weight for the target protein, BhrPET, is 25 kDa (Xi et al. 2021). Since no purification step was performed, multiple protein bands were visible, indicating the presence of both target and non-target proteins in the supernatant, as shown in Fig. 7. A very faint band was observed at the expected molecular weight. However, to confirm the presence of the target protein, further purification is necessary.
The SDS-PAGE analysis revealed multiple protein bands, indicating the presence of both target and non-target proteins. The lack of a purification step contributed to the complexity, making it difficult to definitively identify target proteins due to overlapping bands.
To address this, purification using methods like immobilized metal affinity chromatography (IMAC) could reduce background proteins, aiding in clearer identification of target bands. Western blotting with specific antibodies could also confirm target protein presence, while higher expression levels or more sensitive staining (e.g., silver staining) could improve detection. Concentrating protein samples post-purification might further enhance visibility.
In conclusion, the SDS-PAGE results highlight the need for optimized sample preparation for better identification of target proteins. However, as our focus was on identifying candidates with enzymatic activity for spore display, we concluded that the observed activity was adequate for selection.
References
Jeong H., Jeong D. E., Park S. H., Kim S. J., Choi S. K. (2018): Complete Genome Sequence of Bacillus subtilis Strain WB800N, an Extracellular Protease-Deficient Derivative of Strain 168. Microbiol Resour Announc. 7(18), e01380-18. https://doi.org/10.1128/MRA.01380-18
Kademi A., Aït-Abdelkader N., Fakhreddine L. & Baratti J. (2000): Purification and characterization of a thermostable esterase from the moderate thermophile Bacillus circulans. Journal of microbiology and biotechnology 54, 173–179. https://doi.org/10.1007/s002530000353
Popp P. F., Dotzler M., Radeck J., Bartels J., Mascher T. (2017): The Bacillus BioBrick Box 2.0: expanding the genetic toolbox for the standardized work with Bacillus subtilis. Scientific reports 7(1), 15058. https://doi.org/10.1038/s41598-017-15107-z
Xi X., Ni K., Hao H., Shang Y., Zhao B., Qian Z. (2021): Secretory expression in Bacillus subtilis and biochemical characterization of a highly thermostable polyethylene terephthalate hydrolase from bacterium HR29. Enzyme and microbial technology 143, 109715. https://doi.org/10.1016/j.enzmictec.2020.109715
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