Part:BBa_K5117019
BsRBS-AtBglA
This part serves as translational unit composed of the ribosome binding site of Bacillus subtilis (BBa_K5117000) and the bglA gene of Acetivibrio thermocellus (BBa_K5117009) encoding a β-glucosidase (EC 3.2.1.21).
Biosafety level: S1
Target organism: Bacillus subtilis
Main purpose of use: Testing enzyme functionality in the host B. subtilis
Potential application: Degradation of cellobiose
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 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_K5117009) 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 29.7 ng/µl (digested pSB1C3) and 14.6 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: 277.5 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-AtBglA 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-AtBglA and the integrative expression plasmid pBS2E-xylR-PxylA-AtBglA were verified by sequencing and successfully generated (DNA concentrations: 601.6 ng/µl, 92.2 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 β-glucosidases
We initially tested the β-glucosidases in liquid cultures (see qualitative LB-Agar-Esculin plate assay).
For the expression and testing of heterologously expressed enzymes in B. subtilis, we induced protein production by adding 0.5% xylose once cultures reached an OD600 of 0.5 – 0.6. After 24 hours, cells were harvested and lysed for testing using quantitative assays.
Additionally, samples were 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 AtBglA, as "pBS0EX-AtBglA." Similarly, B. subtilis WB800N transformed with the integrative plasmid pBS2E-xylR-PxylA containing the same enzyme will be referred to as "pBS2EX-AtBglA."
LB-Agar-Esculin plates for determination of β-glucosidase activity
LB-Agar-Esculin plates (see recipe on Experiments page), with and without inducer (0.5 % xylose), were used to test whether our generated intracellular β-glucosidase could degrade esculin as an alternative substrate for cellobiose, based on the observation of black halo formation.
Overnight cultures were grown (180 rpm, 37 °C), adjusted to an OD600 of 0.5 the next day, and 10 µL of each culture was added to the plates, which were then incubated for 24 hours at 37 °C (as shown in Fig. 6). Both B. subtilis clones, generated during the cloning procedure, were tested; however, results for the strain containing the integrative vector (pBS2EX) are not shown, as no other results were observed.
The assay showed black halo formation around the positive control (PC, Accellerase® 1500), the negative control (NC, WB800N culture), and our β-glucosidase cultures, indicating esculin degradation in all samples. No increased activity was observed on plates with xylose, suggesting that the expected enzyme induction did not lead to detectable differences, likely due to high background activity. The similar halo formation seen with the PC further suggested background activity from native B. subtilis enzymes capable of degrading esculin. Due to these limitations, the assay was discontinued.
Determination of glucose produced from cellobiose degradation
Due to the high background activity observed in the previous assay (LB-Agar-Esculin plates), we decided to quantify the glucose produced from the degradation of cellobiose because of the induced expression of our glucosidases. To perform the reaction with cellobiose, intracellular enzymes were accessed by lysing the cells. Detailed procedures are available on the Experiments page.
The glucose concentration in the reaction was determined using the Amplex™ Red Glucose/Glucose Oxidase Assay Kit. This assay involved preparing a working solution containing Amplex® Red reagent, horseradish peroxidase (HRP), and glucose oxidase, which was then mixed with glucose standards and samples. A more detailed protocol can be found on the Experiments page. A glucose calibration curve was generated using standard concentrations ranging from 0 µM to 150 µM (see Results page, glucose assay).
The lysate of pBS0EX-AtBglA was incubated with 50 mM cellobiose (diluted in 1X reaction buffer) at 50 °C for up to 24 hours, followed by termination of the reaction. The glucose produced from cellobiose degradation was measured at 560 nm using the glucose assay to evaluate enzyme performance (see Experiments page), as shown in Fig. 7.
The results indicate that the induced expression (0.5 % xylose was used as an inducer) of pBS0EX-BhBglA ( BBa_K5117017), produced more glucose compared to its control and other enzymes (pBS0EX-AtBglA ( BBa_K5117019) and pBS0EX-PpBglB ( BBa_K5117018), suggesting high enzymatic activity. pBS0EX-AtBglA ( BBa_K5117019) showed low activity, with slightly increased glucose production in the induced state compared to the control. Interestingly, pBS0EX-PpBglB ( BBa_K5117018) exhibited higher absorbance in the control sample compared to the induced state, suggesting unexpected glucose production without induction.
The green bar, representing the cellobiose standard absorbance at 560 nm, suggests the presence of impurities in the cellobiose, resulting in high background signal of the substrate. Furthermore, the lower absorbance values of the enzyme samples compared to the substrate indicate potential interference from the lysate with the glucose assay's coupled reaction, making the assay unsuitable for accurately testing lysate samples.
pNPG assay for β-glucosidase activity determination
Due to these unexpected results and potential interference from the lysate, we decided to use commercially available p-nitrophenyl-β-D-glucopyranoside (pNPG) as an alternative substrate to analyze the β-glucosidase activity.
The activity of β-glucosidase was determined by measuring the hydrolysis of pNPG, using the initial rate of accumulation of the colored product, p-nitrophenol (pNP), following the method of by Korotkova et al. (2009) (see Experiments page). For activity assessment, 20 µL of β-glucosidase lysate was mixed with 180 µL of 5 mM pNPG substrate, dissolved in 50 mM sodium phosphate buffer (pH 7.0) and incubated at 50 °C for 10 minutes. The reaction was stopped by adding 100 µL of ice-cold 0.5 M Na2CO3, allowing for subsequent measurement of pNP formation at 405 nm. The results are shown in Fig. 8. A negative control, without lysate, was included to determine the background signal of the substrate, and the control values were subtracted from the obtained results to reflect actual enzyme activity.
The induced sample of pBS0EX-BhBglA ( BBa_K5117017) shows a higher absorbance compared to the control, indicating β-glucosidase activity. pBS0EX-AtBglA/pBS2EX-AtBglA ( BBa_K5117019) and pBS0EX-PpBglB/ pBS2EX-PpBglB ( BBa_K5117018), as well as pBS2EX-BhBglA the show comparable absorbance between induced and control samples, indicating minimal or no increase in activity upon induction. This could be due to insufficient enzyme yield during cell lysis, leading to reduced detectable activity. The negative control, WB800N, shows similar low absorbance for both conditions, confirming the absence of enzyme activity.
The results suggest that pBS0EX-BhBglA ( BBa_K5117017) in the induced state exhibits higher enzyme activity (approx. 10-fold), while other samples display almost no or minimal increase, indicating lower or absent induction effects or a lack of enzyme activity towards the substrate. Additionally, the enzyme activity in pBS0EX-BhBglA is higher compared to pBS2EX-BhBglA, likely due to the replicative nature of pBS0EX resulting in higher expression compared to the integrative vector (pBS2EX).
Based on the results shown in Fig. 7 and Fig. 8, BhBglA ( BBa_K5117017) demonstrated the highest activity, particularly among the replicate vector (pBS0EX strains), in both the glucose and pNPG assays, establishing it as the most promising candidate out of three. Therefore, BhBglA ( BBa_K5117017) was selected for enzyme display on spores. Additionally, due to challenges associated with testing lysate, PpBglB ( BBa_K5117018) was also chosen for spore display, supported by favorable findings from literature, despite no activity being observed in the assays.
Determination of molecular weight
To determine the molecular weight of the expressed proteins, a 10 % SDS-PAGE was performed for the β-glucosidases. 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 our β-glucosidase AtBglA we used the pBS0EX-AtBglA strain. The expected molecular weight for the target protein, AtBglA, is 51 – 52 kDa. Since no purification step was performed, multiple protein bands are visible, indicating the presence of both target and non-target proteins in the supernatant, as shown in Fig. 9. As enzyme purification was not prioritized, and we applied cell lysate directly onto the gel, the presence of multiple protein bands was expected. These bands represent both the target enzymes and other cellular proteins. Consequently, the gel displays several protein bands, which makes it challenging to identify the specific band corresponding to the expressed β-glucosidase gene.
We also analyzed the debris from β-glucosidase expression, as shown in Fig. 10. Like the results obtained from the cell lysate, multiple bands were observed on the gel, making it difficult to accurately identify the target protein bands based on molecular weight. The presence of these overlapping bands further complicates the identification of the specific β-glucosidase proteins.
The SDS-PAGE analysis of endoglucanases 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
Korotkova, O. G., Semenova, M. V., Morozova, V. V., Zorov, I. N., Sokolova, L. M., Bubnova, T. M., Okunev, O. N., Sinitsyn, A. P. (2009). Isolation and properties of fungal beta-glucosidases. Biochemistry. Biokhimiia, 74(5), 569–577. https://doi.org/10.1134/S0006297909050137
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
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