Part:BBa_K5117017
BsRBS-BhBglA
This part serves as translational unit composed of the ribosome binding site of Bacillus subtilis (BBa_K5117000) and the bglA gene of Bacillus halodurans (BBa_K5117007) 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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 579
- 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_K5117007) 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 13.2 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: 252.7 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. Either, the vector backbone was amplified after isolation, resulting in DNA concentrations of 113.3 ng/µl pBS0E-xylR-PxylA or isolated from E. coli DH10β, resulting in DNA concentrations of 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-BhBglA 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-BhBglA and the integrative expression plasmid pBS2E-xylR-PxylA-BhBglA were verified by sequencing and successfully generated (DNA concentrations: 405.8 ng/µl, 147.8 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 BhBglA, as "pBS0EX-BhBglA." Similarly, B. subtilis WB800N transformed with the integrative plasmid pBS2E-xylR-PxylA containing the same enzyme will be referred to as "pBS2EX-BhBglA."
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 (Ouyang et al. 2023).
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 (Thermo Fisher Scientific Inc., United States). 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-BhBglA 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/pBS2EX-AtBglA ( BBa_K5117019) and pBS0EX/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.
Engineering success
Within the framework of the iGEM competition 2024, our work on β-glucosidase activity assays faced challenges, particularly with background interference and substrate suitability. Initial attempts using LB-Agar-Esculin plates and cellobiose degradation assays highlighted the complexities associated with substrate specificity and lysate interference, respectively. By exploring alternative substrates like pNPG and optimizing assay conditions, we improved the reliability and accuracy of our measurements. Detailed information regarding all tested β-glucosidases can be reviewed on the Engineering page (TU Dresden 2024 iGEM Team).
Engineering cycles during the development of a β-glucosidase activity assay
During the evaluation of β-glucosidase enzymatic activity, we discovered that our initial assay did not perform as intended. Therefore, we went through rounds of the design, build, test and learn stages. The section below provides information on how we addressed the challenges encountered during the development of both qualitative and quantitative assays for β-glucosidase activity determination.
Design
Initially, we decided to test the activity of the intracellularly expressed β-glucosidases (including BhBglA) on LB-Agar-Esculin plates, aiming to observe black halo formation around the colonies as an indicator of enzyme activity.
Build
Overnight cultures of our strains were prepared, and the next day, 10 µl of OD600 = 0.5 culture was spotted onto plates (with and without 0.5 % xylose inducer). E. coli and WB800N strains were used as controls. The plates were incubated overnight, and halo formation was monitored and documented every hour for up to 6 hours, and again at 24 hours.
Test
No halo formation was observed before 6 hours, and after 24 hours, black halos were formed around every colony, regardless of induction (Fig. 6). This suggested that even if β-glucosidase activity existed, it was obscured by high background activity.
Learn
We learned that either the background activity was too high, preventing detection of β-glucosidase activity, or that our selected enzymes were not capable of degrading esculin effectively. The high background potentially resulted from aryl-β-glucosidases, which are natively produced in B. subtilis. These enzymes can specifically degrade aryl-glucosides, but are not able to cleave cellobiose (Ouyang et al. 2023).
Design
Due to the high background activity observed in the previous assay (LB-Agar-Esculin plates), we decided to quantify the glucose produced from cellobiose degradation due to the induced expression of our β-glucosidases.
Build
To access the intracellular enzymes, we lysed the cells prior to the reaction with cellobiose instead of using liquid cultures. The glucose concentration in the reactions was determined using the Amplex™ Red Glucose/Glucose Oxidase Assay Kit.
Test
The lysate of pBS0EX-BhBglA was incubated with 50 mM cellobiose at 50 °C for up to 24 hours, after which the reaction was terminated. Glucose production from cellobiose degradation was measured at 560 nm using the glucose assay to evaluate enzyme performance (Fig. 7). The results indicated that induced expression (0.5 % xylose) of pBS0EX-BhBglA produced more glucose compared to its control and the other enzymes, indicating high enzymatic activity. The high absorbance of the cellobiose standard suggested the presence of impurities (e.g., glucose), resulting in a high background signal.
Learn
The high absorbance of the substrate suggested potential lysate interference, rendering the assay unsuitable for accurately quantifying the glucose in the applied samples. We learned that interference from the lysate with the glucose assay's coupled reaction made this approach unsuitable for accurate testing.
Design
In this iteration, our goal was to determine the maximum lysate concentration that could be used without interfering with the glucose assay's coupled reaction. We aimed to optimize the lysate concentration in order to minimize interference while still retaining sufficient β-glucosidase activity for accurate measurement.
Build
The assay was set up using varying amounts of lysate from WB800N, serving two purposes: the culture contained 0.5 % xylose during the expression phase to evaluate any impact on glucose levels, and to ensure no β-glucosidase activity from heterologously expressed enzymes, thereby providing a baseline for comparison with our enzymes.
Test
The lysate (at varying percentages) was mixed with 50 mM cellobiose, 200 µM glucose, and 1X reaction buffer from the Amplex™ Red Glucose/Glucose Oxidase Assay Kit. Glucose concentration was measured and absorbance was recorded at 560 nm. The reduction of the lysate volume decreased background interference, with the aim of consistently detecting 200 µM glucose (see Engineering page for respective data).
Learn
We learned that optimizing lysate concentration is crucial for minimizing interference from the lysate. After diluting the reaction with the glucose working solution, we determined that the effective lysate concentration should be a maximum of 0.5 % in reactions involving the glucose assay. However, even at 0.5 % lysate, a decrease in glucose detection was observed, indicating some residual interference. (Note: This value effectively doubles due to the dilution factor introduced by the glucose assay working solution.) Therefore, in reactions with cellobiose, we decided to use up to 1% lysate in the reaction mixture to achieve reliable results, balancing enzyme activity and interference.
Design
In this iteration, we aimed to evaluate the β-glucosidase activity of our enzymes over a prolonged incubation period. The goal was to determine glucose production from cellobiose degradation using an optimized lysate concentration that minimized interference.
Build
Reactions were set up with the β-glucosidases (including pBS0EX-BhBglA and pBS2EX-BhBglA) for 24 hours at 50 °C, using a maximum lysate concentration of 1 % with 50 mM cellobiose. After incubation, the glucose concentration was determined using the Amplex™ Red Glucose/Glucose Oxidase Assay Kit.
Test
Reducing the lysate concentration led to decreased enzyme activity, resulting in lower glucose production. No difference was observed between the induced and control samples, and in some cases, the control even showed higher absorbance than the induced sample. However, both were still higher than the cellobiose-only control (see Engineering page for respective data).
Learn
We observed a high background signal from cellobiose, possibly indicating impurities or inhibition of glucose detection. Additionally, reducing the lysate concentration may have led to some loss of enzyme activity, impacting overall glucose production.
Design
To address the high interference observed in the previous iteration, we aimed to purify the cellobiose to reduce interference in glucose detection.
Build
The cellobiose substrate was purified by treating it with glucose oxidase at 37 °C for 3 hours. The reaction was then stopped by heating to 85 °C, and impurities were removed by filtration before using the substrate in a follow-up reaction with lysate.
Test
The glucose assay with the purified cellobiose showed no improvement, as the background signal remained. Additionally, the H2O2 produced from the reaction between glucose and glucose oxidase interfered with resorufin, the color-changing substrate in the glucose assay kit.
Learn
Purification of the cellobiose substrate did not reduce the background signal. We concluded that using an alternative coupled assay to determine the concentration of glucose might be useful. Another option would be to use a commercial chromogenic substrate, such as pNPG.
Design
To overcome the interference issues observed with cellobiose, we decided to use pNPG as the substrate for the β-glucosidase activity assay, as it provides a direct colorimetric measurement.
Build
The reaction was set up with 5 mM pNPG instead of cellobiose, and the absorbance was measured at 405 nm to quantify β-glucosidase activity.
Test
The β-glucosidase activity of pBS0EX-AtBglA and pBS2EX-BhBglA was determined using pNPG as the substrate. Absorbance at 405 nm was measured, indicating the formation of p-nitrophenol (pNP) from pNPG degradation by the β-glucosidase (Fig. 8).
Learn
Using pNPG as a substrate effectively eliminated the background interference issue, providing a more reliable and straightforward measurement of β-glucosidase activity. This approach proved to be a suitable alternative for assessing enzyme performance, especially when using unpurified lysate.
Conclusion
The evaluation of β-glucosidase activity faced several challenges. Initial assays using LB-Agar-Esculin plates showed high background activity, possibly due to endogenous activity from B. subtilis or ineffective enzyme-substrate interactions. When using cell lysates, interference in glucose assays and substrate degradation caused unreliable results. Reducing lysate concentration improved accuracy but decreased enzyme activity. Efforts to purify the cellobiose substrate and mitigate interference were unsuccessful. Switching to the substrate pNPG solved these issues, offering a clearer and more reliable detection of β-glucosidase activity.
Despite initial challenges during the development of enzyme activity assays, we successfully demonstrated the promise of our approach by going through multiple iterations of design, build, test and learn stages. Finally, we achieved engineering success with regard to the investiged β-glucosidases, as the part BsRBS-BhBglA, documented on this page, demonstrated high activity in pNPG assays (compared to the control as well as other tested β-glucosidases). However, pNPG was not the target substrate. Therefore, future experiments must consider substrate specificity to ensure optimal enzyme selection.
Nevertheless, BhBglA is a promising candidate for the final application of degrading cellobiose and was therefore selected for enzyme immobilization on B. subtilis spores (see composite parts BBa_K5117038 , BBa_K5117039 , BBa_K5117040 ), representing the main goal of the TU Dresden 2024 iGEM Team.
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 BhBglA we used the pBS0EX-BhBglA strain. The expected molecular weight for the target protein, BhBglA, is 51 – 52 kDa (Naz et al. 2010). 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 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
Naz S., Ikram N., Rajoka M. I., Sadaf S., Akhtar M. W. (2010): Enhanced production and characterization of a β-glucosidase from Bacillus halodurans expressed in Escherichia coli. Biochemistry (Moscow) 75, 513-518. https://doi.org/10.1134/s0006297910040164
Ouyang B., Wang G., Zhang N., Zuo J., Huang Y., Zhao X. (2023): Recent Advances in β-Glucosidase Sequence and Structure Engineering. Molecules 28(13). https://doi.org/10.3390/molecules28134990
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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