Part:BBa_K5117011

BsRBS-BsEglS

This part serves as translational unit composed of the ribosome binding site of Bacillus subtilis (BBa_K5117000) and the eglS gene of Bacillus subtilis (BBa_K5117001) encoding an endoglucanase (EC 3.2.1.4).

Biosafety level: S1

Target organism: Bacillus subtilis

Main purpose of use: Testing enzyme functionality in the host B. subtilis

Potential application: Degradation of cellulose

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 625
  
• 1000
  COMPATIBLE 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_K5117001) 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 17.2 ng/µl.

Fig. 1: DNA Ladder (A) and agarose gel electrophoresis of pSB1C3 Backbone PCR (B). A: 1 kb Plus DNA Ladder from New England Biolabs (NEB). B: Backbone PCR of pSB1C3. Oligonucleotides for amplification can be found on the Experiments page. The correct PCR product has a size of 2043 bp. DNA fragments of other sizes represent unspecific bands. 1 kb Plus DNA Ladder (NEB) served as marker (M). The pSB1C3 backbone was purified by gel extraction resulting in a DNA concentration of 190.2 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).

Fig. 2: Agarose gel electrophoresis: Insert amplification of pSB1C3-BsEglS by Colony PCR of transformed E. coli DH10β cells. Oligonucleotides for amplification can be found on the Experiments page. Numbers 1-4 correspond to chosen colonies. The correct PCR product has a size of 1829 bp. The negative control displayed no band, but was loaded onto another gel and is therefore not shown here. 1 kb Plus DNA Ladder (NEB) served as marker (M). Colony 1 resulted in the highest plasmid concentration and was subsequently verified by sequencing revealing the correct insert sequence.

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: 297.6 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-P_xylA) and integrative (pBS2E-xylR-P_xylA) 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 151.9 ng/µl pBS0E-xylR-P_xylA and 127.6 ng/µl pBS2E-xylR-P_xylA. These vectors were digested with EcoRI and PstI (Fig. 3) and purified via gel extraction using the HiYield® PCR Clean-up/Gel Extraction Kit (SLG, Germany).

Fig. 3: Agarose gel electrophoresis: Restriction (Res) of expression vectors pBS0E-xylR-P_xylA (pBS0EX) and pBS2E-xylR-P_xylA (pBS2EX) with EcoRI and PstI. The digested plasmid backbones have sizes of 8114 bp and 7758 bp, respectively. Strong bands at approximately 9000 bp could represent undigested plasmids as well as digested ones due to high size inaccuracy of large bands. Weak bands at 1102 bp represent the RFP insert being cut out of the vector. Large bands were purified by gel extraction and resulted in 17.9 ng/µl and 31.3 ng/µl DNA for the digested vectors pBS0EX and pBS2EX, respectively. 1 kb Plus DNA Ladder (NEB) served as marker (M).

The enzyme part was amplified via PCR (Fig. 4) using the plasmid template pSB1C3-BsEglS and subsequently digested and purified via PCR clean up.

Fig. 4: Agarose gel electrophoresis: PCR of part BsEglS. Oligonucleotides for amplification can be found on the Experiments page. The correct PCR product has a size of 1566 bp. The larger band probably represents the plasmid pSB1C3-BsEglS used as template. 1 kb Plus DNA Ladder (NEB) served as marker (M). BsEglS was purified by gel extraction resulting in a DNA concentration of 154.1 ng/µl.

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. 5). Colonies with a band at the correct size of the insert were chosen for plasmid isolation. Finally, the replicative expression plasmid pBS0E-xylR-P_xylA-BsEglS and the integrative expression plasmid pBS2E-xylR-P_xylA-BsEglS were verified by sequencing and successfully generated (DNA concentrations: 601.3 ng/µl, 93.6 ng/µl).

Fig. 5: Agarose gel electrophoresis: Insert amplification of pBS0E-xylR-P_xylA-BsEglS (pBS0EX-BsEglS) and pBS2E-xylR-P_xylA-BsEglS (pBS2EX-BsEglS) by Colony PCR of transformed E. coli DH10β cells. Oligonucleotides for amplification can be found on the Experiments page. Numbers 1-8 correspond to chosen colonies. Correct PCR products have a size of 1717 bp for pBS0EX-BsEglS and 1918 bp for pBS2EX-BsEglS. Negative controls (NC) displayed no band. Whereas the NC for pBS0EX is shown here, the NC for pBS2EX was loaded onto another gel and is therefore not depicted. 1 kb Plus DNA Ladder (NEB) served as marker (M). Colony 2 of pBS0EX-BsEglS and colony 6 of pBS2EX-BsEglS were verified by sequencing and contained the correct insert sequence.

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. 6). Two colonies each with the correct insert size were chosen for cryo-conservation, serving as biological duplicates.

Fig. 6: Agarose gel electrophoresis: Insert amplification of pBS0E-xylR-P_xylA-BsEglS (pBS0EX-BsEglS) and pBS2E-xylR-P_xylA-BsEglS (pBS2EX-BsEglS) by Colony PCR of transformed B. subtilis WB800N cells. Oligonucleotides for amplification can be found on the Experiments page. Numbers 1-4 correspond to chosen colonies. 1 kb Plus DNA Ladder (NEB) served as marker (M). pBS0EX: Primer pair 1 was used for the amplification of a small fragment including BsEglS only (1566 bp). Primer pair 2 was used for amplification of a large fragment including xylR-P_xylA-BsEglS (3197 bp). Colonies 1 and 3 of pBS0EX-BsEglS were correct and chosen for cryo-conservation. pBS2EX: Primer pair 3 was used to check downstream integration by amplification of a fragment including BsEglS and ‘lacA (2104 bp). Primer pair 4 was used to check upstream integration by amplification of a fragment including lacA’ and the erythromycin resistance gene erm (1370 bp).Colonies 2 and 3 of pBS2EX-BsEglS were correct and chosen for cryo-conservation. Negative controls of all primer pairs (NC 1-4) displayed no bands.

Expression of endoglucanases

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 endoglucanases. 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-P_xylA containing one of our enzymes, such as BsEglS, as "pBS0EX-BsEglS." Similarly, B. subtilis WB800N transformed with the integrative plasmid pBS2E-xylR-P_xylA containing the same enzyme will be referred to as "pBS2EX-BsEglS."

CMCase activity determination

To assess the CMCase activity of heterologously expressed endoglucanase BsEglS in B. subtilis, we initially performed a qualitative assay on 1%-Carboxymethyl cellulose (CMC)-Agar plate (see the Experiments page). We decided to test pBS0EX-BsEglS (replicative vector), assuming it would exhibit higher activity than pBS2EX-BsEglS (integrative vector).

Following the expression of BsEglS, induced with 0.5% xylose, we applied 15 µl of the supernatant to wells at the center of 1%-CMC-agar plates. The plates were incubated at 50 °C for 24 hours, after which we stained them using the congo red method to visualize enzyme activity, as shown in Fig. 7. The control strain WB800N and cultures without the addition of inducer were used as controls. Halo formation, indicating CMC degradation, was used to determine enzyme activity.

Negligible halos were observed on the plate of WB800N, suggesting only basal endoglucanase activity in the control strain, confirming the presence of the eglS gene in B. subtilis. Similarly, negligible halo formation was observed in the uninduced culture, indicating minimal endoglucanase expression without induction, with only basal enzyme activity from eglS being present.

In contrast, clear halos were observed for pBS0EX-BsEglS induced with xylose, indicating successful expression and activity of the endoglucanase compared to both the WB800N control and the uninduced culture. This demonstrates that the enzyme is effectively capable of degrading CMC.

Fig. 7: Qualitative CMCase activity assay of supernatant from pBS0EX-BsEglS on 1%-CMC-Agar plates. To determine CMCase activity, 15 µL of the supernatant was applied to 1%-CMC-Agar plates. After incubation at 50 °C for 24 hours, then stained with congo red and destained with 1 M NaCl to visualize enzyme activity. Clear halos around the wells indicate CMC degradation by the expressed endoglucanase. Uninduced culture of pBS0EX-BsEglS and the strain WB800N were used as controls (see Experiments page).

Determining endoglucanase activity using discontinuous DNS assay

To decide whether BsEglS (BBa_K5117011) or BpEglA ( BBa_K5117012) should be used for further immobilization on the spore surface, a discontinuous 3,5-dinitrosalicylic acid (DNS) assay (see Experiments page) was performed which is typically applied to estimate the amount of the reducing ends produced during the CMC degradation (Bailey et al. 1981). For this purpose a calibration curve was generated using calibration standards with glucose concentrations ranging from 200 μg/ml to 2000 μg/ml (see Results page, DNS assay).

For the DNS assay, supernatants of pBS0EX-BsEglS and pBS0EX-BglA were used. The reaction was conducted with CMC diluted in 50 mM phosphate buffer (pH 7) at 50 °C for different time intervals (1 hour, 5 hours and 13 hours). DNS stop solution was then applied as described on the Experiments page, and the absorbance was measured at 540 nm. Controls included supernatants from B. subtilis WB800N, and supernatants from cultures without the addition of a protein expression inducer. Results are shown in Fig. 8.

Fig. 8: Discontinuous DNS assay for determining endoglucanase activity of pBS0EX-BsEglS and pBS0EX-BsEglA. The assay was conducted in 50 mM phosphate buffer (pH 7) at 50 °C, with 0.93% CMC as substrate. Absorbance was measured at 540 nm. Supernatants from non-induced strains, as well as the WB800N strain, were used as controls. Protein expression was induced by adding 0.5% xylose during the exponential growth phase. To account for background signal, the absorbance from a negative control containing only CMC in buffer was subtracted from the measured values (see Experimentspage). The measurement was conducted in a single biological replicate (N = 1).

Since B. subtilis naturally produces BsEglS, a notable background activity was detected after 13 hours of reaction, as previously shown with the qualitative CMC assay. However, this background activity did not interfere with the assessment of recombinant protein activity. The amount of reducing ends produced in samples from uninduced cultures containing plasmids with BsEglS and BpEglA genes was comparable to the WB800N control, reaching 161.68 μg/ml and 151.68 μg/ml, respectively, after 13 hours. Supernatants containing BpEglA led to the formation of 360.00 μg/ml reducing ends, while BsEglS-containing supernatants showed the highest activity, correlating with 500.00 μg/ml of reducing ends produced in 13 hours. Therefore, BsEglS was selected for further spore immobilization experiments.

Noteworthy, since the experiments were carried out with unpurified endoglucanases, the activity values were not standardized to the amount of enzyme responsible for the reaction, which led to limited comparability of the results. It is possible that BsEglS appears more active than BpEglA due to being produced in larger quantities by B. subtilis WB800N under the test conditions rather than having inherently higher activity. Additionally, the chosen discontinuous assay does not reflect the initial velocity of enzyme catalysis and could be influenced by factors such as thermostability. Despite these considerations, BsEglS remained the candidate for spore immobilization experiments, as it is naturally produced by B. subtilis, which may enhance its chances for successful production on the spore surface of this organism.

Determination of molecular weight

To determine the molecular weight of the expressed proteins, a 10% SDS-PAGE was performed for the endoglucanases. 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.

We focused on BsEglS (BBa_K5117011) and BpEglA ( BBa_K5117012) in the SDS-PAGE analysis, as shown in Fig. 9. Distinct bands were observed in the induced culture lanes, corresponding to 55 kDa for BsEglS (predicted, see https://www.uniprot.org/uniprotkb/P10475/entry) and 72 kDa for BpEglA (Lima et al. 2005), verifying the successful expression of these proteins. However, since BsEglS is naturally produced by B. subtilis, faint bands were also present in all samples, indicating some basal expression of this enzyme even without induction.

Fig. 9: SDS-PAGE analysis (see Experimentspage) of endoglucanase supernatants (pBS0EX-BpEglA and pBS0EX-BsEglS) on a 10 % gel. The figure shows supernatant samples from induced (+) and uninduced (-) cultures. 4 µl of the PageRuler™ Plus Prestained Protein Ladder (10 to 250 kDa) (M) were used to estimate molecular weight, with the WB800N strain serving as a negative control. Each supernatant(45 µL) was mixed with 15 µL of 4X loading buffer, heated at 95 °C for 10 minutes, and 20 µL of each sample were loaded onto the gel. The gel was stained with Coomassie Blue and destained to visualize protein bands. The expected molecular weights for the target proteins are 72 kDa for BpEglA and 55 kDa for BsEglS. Multiple protein bands are visible, indicating the presence of both target and non-target proteins in the supernatant.

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

Bailey, M. J., & Nevalainen, K. M. H. (1981). Induction, isolation and testing of stable Trichoderma reesei mutants with improved production of solubilizing cellulase. Enzyme and Microbial Technology, 3(2), 153–157. https://doi.org/10.1016/0141-0229(81)90076-4

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

Lima A. O., Quecine M. C., Fungaro M. H., Andreote F. D., Maccheroni W., Araújo W. L., Silva-Filho M. C., Pizzirani-Kleiner A. A., Azevedo J. L. (2005): Molecular characterization of a β-1, 4-endoglucanase from an endophytic Bacillus pumilus strain. Applied microbiology and biotechnology 68, 57-65. https://doi.org/10.1007/s00253-004-1740-1

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