Part:BBa_K5117043

PcotYZ-BsRBS-PpBglB-L3-CotY-B0014

This part serves as transcriptional unit composed of:

• promoter PcotYZ of Bacillus subtilis (BBa_K5117021)
• ribosome binding site of Bacillus subtilis (BBa_K5117000)
• bglB gene of Paenibacillus polymyxa encoding a β-glucosidase (EC 3.2.1.21),
addition of a rigid linker (L3) downstream of the coding sequence encoding the amino acids GGGEAAAKGGG (BBa_K5117031)
• cotY gene of Bacillus subtilis (BBa_K5117022)
• bidirectional terminator B0014 (BBa_B0014)

Biosafety level: S1

Target organism: Bacillus subtilis

Main purpose of use: Immobilization of PpBglB on the spore crust of B. subtilis (spore surface display)

Application: Degradation of cellobiose

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 NgoMIV site found at 1600
  
• 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_K5117031) of the enzyme.

Construct design

For compatibility with the BioBrick RFC[10] standard, the restriction sites EcoRI, 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).

To express target genes only under sporulation, the sporulation-dependent promoter P_cotYZ of B. subtilis was chosen. In previous studies, this promoter has so far provided the highest activity for spore surface display (Bartels et al. 2018, unpublished data of Elif Öztel). The promoter was followed by the ribosome binding site (RBS) for the host B. subtilis with a 7 bp spacer.

To anchor the target enzyme on the spore surface, it was fused to the N-terminus of the anchor protein CotY. This anchor is located in the crust, the outermost spore layer, and has been shown to be well suited for protein immobilization (McKenney et al. 2013, Bartels et al. 2018, Lin et al. 2020).

Moreover, different linkers between the fused target enzyme and anchor protein were analyzed, as these proteins may affect the folding and stability of each other and, eventually, lead to misfolding and reduced activity. Whereas flexible linkers promote the movement of joined proteins and are usually composed of small amino acids (e.g. Gly, Ser, Thr), rigid linkers are usually applied to maintain a fixed distance between the domains (Chen et al. 2013).

Within the framework of the TU Dresden iGEM 2024 Team, three linkers have been tested: 1) A short flexible GA linker (L1) encoding the small amino acids Gly and Ala, 2) A long flexible linker (GGGGS)₄ (L2) which is one of the most common flexible linkers consisting of Gly and Ser residues and 3) A rigid linker GGGEAAAKGGG (L3) in which the EAAAK motif results in the formation of an alpha helix providing high stability (Chen et al. 2013).

The composite part documented in this page contains the rigid linker GGGEAAAKGGG.

Following the CDS of cotY, a spacer consisting of 10 bp of the natural genome sequence downstream from the cotYZ operon was inserted. This creates space before the terminator and ensures that the ribosome is able to read the full length of the CDS. The construct ends with the terminator B0014, a bidirectional terminator consisting of B0012 and B0011.

The entire construct was flanked with the BioBrick prefix and suffix, allowing for cloning via the BioBrick assembly standard and restriction-ligation-cloning. The vector pBS1C from the Bacillus BioBrickBox was used as an integrative plasmid backbone enabling genomic integration into the amyE locus of B. subtilis (Radeck et al. 2013).

Construction of spore display plasmids

First, all biological parts including the enzyme candidate (Fig. 1) as well as the promoter P_cotYZ, the terminator B0014 and the anchor gene cotY (Fig. 2) were amplified by PCR and purified using the HiYield® PCR Clean-up/Gel Extraction Kit (SLG, Germany). The RBS was added by oligonucleotides. The plasmid pSB1C3-PpBglB generated in the subcloning phase served as template for PCR of the enzyme candidate (see BBa_K5117018). The linker was added by oligonucleotides. The promoter and anchor gene were amplified from genomic DNA of B. subtilis W168 and the terminator from a plasmid provided by the laboratory collection of Prof. Thorsten Mascher (General Microbiology, TU Dresden).

Fig. 1: Agarose gel electrophoresis: PCR of PpBglB-L3. Oligonucleotides for amplification can be found on the Experiments page. The correct PCR product has a size of 1424 bp. 1 kb Plus DNA Ladder (NEB) served as marker (M). The PCR product was purified by gel extraction resulting in a DNA concentration of 134.8 ng/µl.

Fig. 2: Agarose gel electrophoresis: PCR of P_cotYZ, cotY and B0014. Oligonucleotides for amplification can be found on the Experiments page. Amplification of P_cotYZ results in a band of 238 bp, L3-cotY displays a band of 514 bp and B0014 140 bp. 1 kb Plus DNA Ladder (NEB) served as marker (M). PCR products were purified resulting in DNA concentrations of 155.7 ng/µl (P_cotYZ), 99.4 ng/µl (L3-cotY), and 52.2 ng/µl (B0014).

The composite part was subsequently assembled via Overlap PCR by complementary overhangs, which were designed and added by oligonucleotides (Fig. 3).

Fig. 3: Agarose gel electrophoresis: Overlap PCR of the spore display construct P_cotYZ-PpBglB-L3-cotY-B0014. Oligonucleotides for amplification can be found on the Experiments page. The correct PCR product has a size of 2241 bp. Other bands are probably caused by unspecific binding of oligonucleotides. 1 kb Plus DNA Ladder (NEB) served as marker (M). The Overlap PCR product was purified by gel extraction resulting in a DNA concentration of 34.3 ng/µl.

Afterwards, the Overlap PCR product was cloned into the vector backbone pBS1C enabling genome integration into the amyE locus in B. subtilis (Radeck et al. 2013). For that purpose, the insert as well as pBS1C were digested with EcoRI and PstI and purified by PCR clean up (DNA concentration: 25.8 ng/µl and 25 ng/µl respectively) using the HiYield® PCR Clean-up/Gel Extraction Kit (SLG, Germany).

After ligation, the plasmid was transformed into chemically competent E. coli DH10β cells and transformants were selected by ampicillin resistance (100 μg/ml ampicillin) encoded on the vector backbone. As the vector was purified via PCR clean up, the vector backbone might re-ligate with the original RFP insert, but colonies with re-ligated plasmids appear pink on the plate and can therefore be distinguished from correct transformants.

The negative control containing no DNA showed no growth. The positive control containing pBS1C resulted in a pink bacterial lawn. The re-ligation control containing the digested vector pBS1C led to growth of approximately 400 pink and 20 white colonies. The transformation with the spore display plasmid pBS1C-P_cotYZ-PpBglB-L3-cotY-B0014 resulted in ≈ 300-400 colonies, both pink and white ones.

White colonies transformed with the spore display plasmid 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 according to the HiYield® Plasmid Mini DNA Kit (SLG, Germany). The correct insert sequence of the plasmid was verified via sequencing by Microsynth Seqlab GmbH. Consequently, the spore display plasmid pBS1C-P_cotYZ-PpBglB-L3-cotY-B0014 was successfully constructed (DNA concentration: 223.1 ng/µl).

Fig. 4: Agarose gel electrophoresis: Insert amplification of P_cotYZ-PpBglB-L3-cotY-B0014 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 2302 bp. The negative control displayed no bands. 1 kb Plus DNA Ladder (NEB) served as marker (M). Colony 1 was verified by sequencing and contained the correct insert sequence.

Generation of Bacillus subtilis strains

Ultimately, this plasmid was transformed into the B. subtilis wildtype W168. Transformants were selected by chloramphenicol resistance (5 μg/ml) encoded on the pBS1C vector backbone. The negative control containing no DNA showed no growth. The transformation with the spore display plasmid pBS1C-P_cotYZ-PpBglB-L3-cotY-B0014, which was linearized by restriction with BsaI prior to transformation, resulted in ≈ 400-500 white colonies.

The successful genomic integration was verified via starch assay (Fig. 5). Four colonies were transferred onto replica and starch plates. If integration into the amyE locus was successful, the native amylase of Bacillus is not produced correctly, resulting in the organism’s inability to degrade starch. Correct transformants displayed no halo around the cells. The wildtype W168 with a functional amylase was used as a control and produces a clear halo. Two biological duplicates of the engineered B. subtilis strain were chosen for cryo-conservation, with clone 1 being used for subsequent activity tests.

Fig. 5: Starch assay of transformed B. subtilis W168 cells. Numbers 1-4 correspond to chosen colonies. W168 served as control and displayed a bright halo. In contrast, no halo was visible for the W168 strain with the integrated spore display construct.

Spore preparation

Spores were prepared by culturing cells in LB medium with chloramphenicol until they reached the exponential growth phase (OD₆₀₀ of 0.4–0.6). After washing and resuspension in DSM, the culture was incubated at 37 °C for 24 hours to induce sporulation. The cells were then lysed using lysozyme and washed with dH₂O and SDS to remove vegetative cell residues. The spore suspension was adjusted to an OD₆₀₀ of 2 for the glucose assay, pNPG assay, and pNPAc assay to achieve a final OD₆₀₀ of 0.2 in the reaction. Further details are available on the Experimentspage.

Glucose assay for determination of glucose concentration after degradation of cellobiose

We first prepared spores displaying BhBglA, as this enzyme showed promising results in previous assays involving induced expression (see BBa_K5117017). Our aim was to determine whether the glucose assay could effectively measure the glucose concentration resulting from the degradation of 50 mM cellobiose by the immobilized enzymes. The assay was performed according to the protocol described on the Experimentspage, using the Amplex™ Red Glucose/Glucose Oxidase Assay Kit. After a 24-hour incubation period, the glucose assay was carried out, and absorbance was measured at 560 nm. We questioned whether a 24-hour incubation period was beneficial, given the low absorbance observed (see , which suggested that the enzyme activity might be inhibited by the accumulation of glucose in the reaction medium. Therefore, we decided to discontinue the 24-hour incubation and instead assessed enzyme activity over a shorter time frame of 30 minutes, collecting samples at 10-minute intervals (three samples in total). Additionally, we included spores displaying PpBglB-L3 ( BBa_K5117043) alongside spores displaying BhBglA-L2 ( BBa_K5117039) to compare their activity. The glucose assay was performed according to the protocol (see Experimentspage), and the results are presented in Fig. 6.

Fig. 6: Glucose concentration determination following degradation of 50 mM cellobiose by spores displaying β-glucosidases (BhBglA-L2 ( BBa_K5117039) and PpBglB-L3 ( BBa_K5117043)) (see Experimentspage). The reaction with 50 mM cellobiose was incubated for 30 minutes at 50 °C, with samples collected every 10 minutes. After incubation, the glucose concentration was determined using the Amplex™ Red Glucose/Glucose Oxidase Assay Kit, with absorbance measured at 560 nm. Spores from the W168 strain were used as a negative control, and 50 mM cellobiose was used as a substrate control. The amount of spores was adjusted to achieve an OD₆₀₀ of 0.2 in the reaction mixture. The measured value from the cellobiose control was subtracted from the enzyme activity measurements to account for the background signal of unpurified cellobiose.

The W168 control showed no absorbance at any time point, indicating no glucose production and confirming the absence of enzymatic activity in the control spores. BhBglA-L2 ( BBa_K5117039) displayed increasing absorbance over time, peaking at 20 minutes and slightly decreasing at 30 minutes. This suggests effective enzymatic activity, though the slight decrease could be due to substrate saturation or measurement variability, as triplicates were not performed. PpBglB-L3 showed no absorbance, similar to the W168 control, indicating no enzymatic activity under these conditions. These results suggest that BhBglA-L2 effectively degrades cellobiose and produces glucose within the 30-minute incubation period, while PpBglB-L3 shows no detectable activity. The peak absorbance at 20 minutes for BhBglA-L2 likely reflects experimental fluctuations, as no triplicates were performed.

pNPG assay for β-glucosidase activity determination

Based on previous results (see pBS0EX-BhBglA/pBS0EX-BhBglA ( BBa_K5117017) we decided to use p-nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate to validate the findings from the glucose assay. The assay was performed according to the protocol outlined in the Experiments page. We tested spores displaying (BhBglA-L1 ( BBa_K5117038), BhBglA-L2 ( BBa_K5117039), BhBglA-L3 ( BBa_K5117040)) as well as (PpBglB-L1 ( BBa_K5117041) and PpBglB-L3 ( BBa_K5117043) in two biological replicates (N = 2), to assess and compare their enzymatic activities using pNPG as a more accessible substrate. The results are shown in Fig. 10.

Fig. 10: Evaluation of enzymatic activity of spore-displayed β-glucosidases (BhBglA-L1 ( BBa_K5117039), BhBglA-L3 ( BBa_K5117043))) The reaction was conducted at 40 °C for 10 minutes, followed by absorbance measurement at 405 nm to indicate the formation of pNP. Spores of the W168 strain were used as a control, and additional control without spore solution was included. The amount of spores was adjusted to achieve an OD₆₀₀ of 0.2 in the reaction mixture. The assay was performed in two biological replicates (N = 2). The absorbance from this control was subtracted from the measured values to account for background signal.

W168 control shows no absorbance, confirming the absence of enzymatic activity and serving as a baseline for comparison. BhBglA-L1 exhibited the highest absorbance (around 2.2), indicating significant enzymatic activity when pNPG was used as a substrate. BhBglA-L2 showed slightly lower activity compared to BhBglA-L1, with an absorbance of approx. A₄₀₅= 1.8. BhBglA-L3 displayed a high absorbance like BhBglA-L1, suggesting comparable activity between these two linkers. Both PpBglB-L1 and PpBglB-L3 showed no absorbance, indicating no enzymatic activity under the tested conditions.

Overall, these results indicate that BhBglA-L1 and BhBglA-L3 exhibited the highest activity among the tested variants, while PpBglB-L1 and PpBglB-L3 showed no significant activity. The use of pNPG as a substrate effectively demonstrated differences in enzyme performance among the linkers and between the two enzymes.

Based on the results of previous assays, immobilized PpBglB on spores was excluded from further testing due to the lack of observable activity. Therefore, we focused on BhBglA which was immobilized on spores fused with three different linkers (BhBglA-L1 , BhBglA-L2 and BhBglA-L3).

References

Bartels J., López Castellanos S., Radeck J., Mascher T. (2018): Sporobeads: The utilization of the Bacillus subtilis endospore crust as a protein display platform. ACS synthetic biology 7(2), 452-461. https://doi.org/10.1021/acssynbio.7b00285

Chen X., Zaro J. L., Shen, W. C. (2013): Fusion protein linkers: property, design and functionality. Advanced drug delivery reviews 65(10), 1357-1369. https://doi.org/10.1016/j.addr.2012.09.039

McKenney P. T., Driks A., Eichenberger P. (2013): The Bacillus subtilis endospore: assembly and functions of the multilayered coat. Nature Reviews Microbiology 11 (1), 33–44. https://doi.org/10.1038/nrmicro2921

Lin P., Yuan H., Du J., Liu K., Liu H., Wang T. (2020): Progress in research and application development of surface display technology using Bacillus subtilis spores. Applied microbiology and biotechnology 104 (6), 2319–2331. https://doi.org/10.1007/s00253-020-10348-x

Öztel, E. & Mascher T. (2024): unpublished data.

Radeck J., Kraft K., Bartels J., Cikovic T., Dürr F., Emenegger J., Kelterborn S., Sauer C., Fritz G., Gebhard S., Mascher T. (2013): The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. Journal of biological engineering 7, 1-17. https://doi.org/10.1186/1754-1611-7-29

Vellanoweth R. L. & Rabinowitz J. C. (1992): The influence of ribosome‐binding‐site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. Molecular microbiology 6(9), 1105-1114. https://doi.org/10.1111/j.1365-2958.1992.tb01548.x