Part:BBa_K5117015

BsRBS-AtCelO

This part serves as translational unit composed of the ribosome binding site of Bacillus subtilis (BBa_K5117000) and the celO gene of Acetivibrio thermocellus (BBa_K5117005) encoding an exoglucanase (EC 3.2.1.176).

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1092
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 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_K5117005) 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 28.4 ng/µl (digested pSB1C3) and 12.2 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-AtCelO by Colony PCR of transformed E. coli DH10β cells. Oligonucleotides for amplification can be found on the Experiments page. Numbers 1-5 correspond to chosen colonies. The correct PCR product has a size of 2312 bp. The negative control displayed no band. 1 kb Plus DNA Ladder (NEB) served as marker (M). Colony 2 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: 322.7 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-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 112.4 ng/µl pBS0E-xylR-P_xylA and 149.9 ng/µl pBS2E-xylR-P_xylA. 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-AtCelO and subsequently digested and purified via PCR clean up.

Fig. 3: Agarose gel electrophoresis: PCR of part AtCelO. Oligonucleotides for amplification can be found on the Experiments page. The correct PCR product has a size of 2041 bp. The larger band probably represents the plasmid pSB1C3-AtCelO used as template. 1 kb Plus DNA Ladder (NEB) served as marker (M). AtCelO was purified by gel extraction resulting in a DNA concentration of 169.1 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. 4). 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-AtCelO and the integrative expression plasmid pBS2E-xylR-P_xylA-AtCelO were verified by sequencing and successfully generated (DNA concentrations: 266.2 ng/µl, 98.5 ng/µl).

Fig. 4: Agarose gel electrophoresis: Insert amplification of pBS0E-xylR-P_xylA-AtCelO (pBS0EX-AtCelO) and pBS2E-xylR-P_xylA-AtCelO (pBS2EX-AtCelO) by Colony PCR of transformed E. coli DH10β cells. Oligonucleotides for amplification can be found on the Experiments page. Numbers 1-6 correspond to chosen colonies. Correct PCR products have a size of 2200 bp for pBS0EX-AtCelO and 2401 bp for pBS2EX-AtCelO. Negative controls (NC) displayed no band, but were loaded onto another gel and are therefore not shown here. 1 kb Plus DNA Ladder (NEB) served as marker (M). Colony 3 of pBS0EX-AtCelO and colony 2 of pBS2EX-AtCelO 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. 5). Two colonies each with the correct insert size were chosen for cryo-conservation, serving as biological duplicates.

Fig. 5: Agarose gel electrophoresis: Insert amplification of pBS0E-xylR-P_xylA-AtCelO (pBS0EX-AtCelO) and pBS2E-xylR-P_xylA-AtCelO (pBS2EX-AtCelO) 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 AtCelO only (2049 bp). Primer pair 2 was used for amplification of a large fragment including xylR-P_xylA-AtCelO (3680 bp). Colonies 1 and 2 of pBS0EX-AtCelO were correct and chosen for cryo-conservation. pBS2EX: Primer pair 3 was used to check downstream integration by amplification of a fragment including AtCelO and ‘lacA (2587 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 1 and 3 of pBS2EX-AtCelO were correct and chosen for cryo-conservation. Negative controls of all primer pairs displayed no bands, but NC1 and NC2 were loaded onto another gel and are therefore not shown here.

Expression of exoglucanases

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 exoglucanases. 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 AtCelO, as "pBS0EX-AtCelO." Similarly, B. subtilis WB800N transformed with the integrative plasmid pBS2E-xylR-P_xylA containing the same enzyme will be referred to as "pBS2EX-AtCelO."

Detemination of exoglucanase activity with PASC assays and PASA assay

We attempted to determine exoglucanase activity using various methods, including assays with avicel and congo red staining, as well as a cellulose overlay assay. Despite multiple iterations, these approaches were unsuccessful. Ultimately, we established the phosphoric acid swollen avicel (PASA) and phosphoric acid swollen cellulose (PASC) assays, which allowed for clear visualization of exoglucanase activity through the appearance of clearance zones, as shown in Fig. 6, without the need for additional staining (based on work of Percival Zhang et al. 2006)). Details of the assay development can be found on the Engineeringpage.

Fig. 6: PASC and PASA assays for determination of exoglucanase activity. AThe arrow indicates the zone of clearance, which signifies exoglucanase activity. The sample used was 20 µL of commercially available cellobiohydrolase I, and the plates were incubated at 50 °C for 24 hours.

The activity of exoglucanases was tested using qualitative phosphoric acid swollen cellulose (PASC) and phosphoric acid swollen avicel (PASA) assays, as described on the the Experimentspage.

For the detection of exoglucanase activity (exemplified using PASA-Agar), supernatants from induced and non-induced pBS0EX-AtCelO cultures were tested. Unfortunately, no enzyme activity was detected, as shown in Fig. 7. The experiment was repeated using supernatants from cultures induced at 28 °C and 42 °C, but without success (data not shown). Consequently, we decided to proceed with both exoglucanase genes for protein immobilization on the spore display surface.

Fig. 7: Qualitative assay for exoglucanase activity on PASA-Agar. Supernatants containing pBS0EX-AtCelO were added to wells on a plate with PASA agar. A culture in which the inducer was not used served as a control. A culture without inducer served as a control. Protein production was induced with 0.5% xylose. After 24 hours of incubation, a zone of clearance was expected to indicate exoglucanase activity, but none was observed.

Determination of molecular weight

To determine the molecular weight of the expressed proteins, an 8 % SDS-PAGE was performed for the exoglucanases. 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.

Since we did not detect any exoglucanase activity, we further analyzed the insoluble fraction (pellet) alongside the supernatant, as shown in Fig. 8. Due to the lack of purification steps, it was difficult to identify AtCelO and AtCelS, expected at approximately 75 - 86 kDa. Additionally, we attempted to express both enzymes at different temperatures (28 °C, 37 °C, and 42 °C), but no bands or activity were detected in any of the conditions (data not shown for 28 °C and 42 °C).

Fig. 7: SDS-PAGE analysis (see Experimentspage) of exoglucanases (pBS0EX-AtCelO (BBa_K5117015) and pBS0EX-AtCelS (BBa_K5117016)) supernatants and pellets on a 8 % gel. The figure shows supernatant and pellet 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 75 kDa for AtCelO (predicted, see https://www.uniprot.org/uniprotkb/Q9L3J2/entry) and 86 kDa for AtCelS (Kruus et al. 1995). Multiple protein bands are visible, indicating the presence of both target and non-target proteins in the soluble and insoluble fraction.

The SDS-PAGE analysis of exoglucanases 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. Despite not detecting any activity, we proceeded with spore display for both genes.

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

Kruus K., Wang W. K., Ching J., Wu J. H. (1995): Exoglucanase activities of the recombinant Clostridium thermocellum CelS, a major cellulosome component. Journal of bacteriology 177(6), 1641-1644. https://doi.org/10.1128/jb.177.6.1641-1644.1995

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

Percival Zhang, Y. H., Cui, J., Lynd, L. R., & Kuang, L. R. (2006). A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: Evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules, 7(2), 644–648. https://doi.org/10.1021