Part:BBa_K5117037

PcotYZ-BsRBS-BsEglS-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)
• eglS gene of Bacillus subtilis without signal peptide encoding an endoglucanase (EC 3.2.1.4),
addition of a rigid linker (L3) downstream of the coding sequence encoding the amino acids GGGEAAAKGGG (BBa_K5117025)
• 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 BsEglS on the spore crust of B. subtilis (spore surface display)

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 NgoMIV site found at 1669
  Illegal AgeI site found at 726
  
• 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_K5117025) 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studies, this promoter has so far provided the highest activity for spore surface display (Bartels et al. 2018, unpublished data of Elif Öztel).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-BsEglS generated in the subcloning phase served as template for PCR of the enzyme candidate (see BBa_K5117011). 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 BsEglS-L3. Oligonucleotides for amplification can be found on the Experiments page. The correct PCR product has a size of 1493 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 72.5 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 149.1 ng/µl (P_cotYZ), 110.5 ng/µl (L3-cotY), and 27.5 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-BsEglS-L3-cotY-B0014. Oligonucleotides for amplification can be found on the Experiments page. The correct PCR product has a size of 2310 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 62.5 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: 27.4 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-BsEglS-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-BsEglS-L3-cotY-B0014 was successfully constructed (DNA concentration: 208.4 ng/µl).

Fig. 4: Agarose gel electrophoresis: Insert amplification of P_cotYZ-BsEglS-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 2371 bp. Two colonies of the re-ligation control (RC) were tested as well, with RC1 appearing white on the plate containing an unknown insert, and RC2 appearing pink on the plate with a probable RFP insert. The negative control displayed no bands. 1 kb Plus DNA Ladder (NEB) served as marker (M). Colony 3 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-BsEglS-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 2.8 for the quantitative assay to achieve a final OD₆₀₀ of 0.2 in the reaction. For qualitative plate assays, an OD₆₀₀ of 0.2 was used. Further details are available on the Experimentspage.

CMCase activity determination

To determine the CMCase activity of BsEglS displaying spores, we first performed a qualitative assay using Carboxymethyl cellulose (CMC)-Agar plates following the protocol described on the Experimentspage (Sharma et al. 2017). The OD₆₀₀ of the spore solution was adjusted to 0.2, and 15 µL of the suspension was applied onto the CMC-Agar plates. After an incubation period of 24 hours at 50 °C, the plates were stained with congo red solution and subsequently destained with 1 M NaCl to observe halo formation. The enzyme activity was visualized by the appearance of clear halos on the congo red stained agar, indicating areas of CMC degradation. The results are shown in Fig. 6.

In the control plate containing W168 spores, there was no visible activity observed, as expected. This confirmed that the control spores, which do not display the BsEglS enzyme, exhibit no enzyme activity. In contrast, the plates with spores displaying BsEglS linked via different linkers (BsEglS-L1, BsEglS-L2, BsEglS-L3) showed clear halos, demonstrating that enzyme activity was present.

Fig. 6: Qualitative CMCase activity assay of BsEglS displaying spores (BsEglS-L1 ( BBa_K5117038), BsEglS-L2 ( BBa_K5117039) and BsEglS-L3 ( BBa_K5117037) on CMC-agar plates (see Experimentspage). To assess CMCase activity, the OD₆₀₀ of the spore solution was adjusted to 0.2, and 15 µL of the suspension was applied to CMC-agar plates. After incubation at 50°C for 24 hours, plates were stained with congo red and destained using 1 M NaCl to visualize enzyme activity. The clear halos indicate CMC degradation by the BsEglS displaying spores.

Determining endoglucanase activity using discontinuous quantitative DNS assay

To quantitatively compare the enzyme activity of endoglucanases displaying-spores (BsEglS-L1 ( BBa_K5117038), BsEglS-L2 ( BBa_K5117039) and BsEglS-L3 ( BBa_K5117037), we used the 3,5-dinitrosalicylic acid (DNS) assay (Bailey et al. 1981), which allowed us to measure the reducing sugars produced during the enzymatic degradation of carboxymethylated cellulose, providing a more detailed comparison of enzyme activity across the different linker systems.

Initially, DNS assay with BsEglS displaying spores (BsEglS-L1, BsEglS-L2, BsEglS-L3) was performed with CMC as substrate for 24 hours at 50 °C to quantitively compare the influence of different linkers on BsEglS activity. Spores from W168 showed no activity as expected since endoglucanases are not present natively on the surface of B. subtilis spores. Small differences between the corresponding activity values could be detected, as shown in Fig. 7. The resulted concentrations of reducing ends were 582.02 μg/ml, 616.81 μg/ml and 626.35 μg/ml for BsEglS-L1, BsEglS-L2 and BsEglS-L3 respectively.

Fig. 7: DNS assay for determination of endoglucanase activity of BsEglS displaying spores (BsEglS-L1 ( BBa_K5117038), BsEglS-L2 ( BBa_K5117039) and BsEglS-L3 ( BBa_K5117037) (see Experimentspage). DNS assay was performed with BsEglS displaying spores (with final OD₆₀₀ of 0.2 in reaction) in 50 mM phosphate buffer pH 7 at 50 °C for 24 hours. The absorbance was measured at 540 nm. The measured values were background corrected. W168: Spore of B. subtilis W168. BsEglS-L1, BsEglS-L2, BsEglS-L3: Spores of B. subtilis W168 displaying BsEglS fused to CotY spore coat protein via linker L1, L2 and L3, respectively. The assay was performed with one biological replicate (N = 1).

The assay was repeated with the reaction conducted for 30 minutes, as shown in Fig. 8. This time BsEglS-L2 showed the lowest activity which resulted in 141.67 μg/ml of produced reducing ends. BsEglS-L1 and BsEglS-L3 performed similarly, with BsEglS-L1 showing slightly higher activity. In samples with BsEglS-L1 338.33 μg/ml of reducing ends were formed and with BsEglS-L3 285.00 μg/ml.

There are different possible explanations as to why no differences were detected after 24 hours. It is possible that, after a certain period, enzyme activity was inhibited, or the enzyme activity was lost due to low thermostability. Furthermore, the influence of saccharides on the germination of B. subtilis should be studied in the future to determine if vegetative cells are formed from spores during the reaction time. Such germination could reduce the number of spores and, consequently, lower the total enzyme activity in the solution.

Fig. 8: DNS assay for determination of endoglucanase activity of BsEglS displaying spores (BsEglS-L1 ( BBa_K5117038), BsEglS-L2 ( BBa_K5117039) and BsEglS-L3 ( BBa_K5117037) (see Experimentspage). DNS assay was performed with BsEglS displaying spores (with final OD₆₀₀ of 0.2 in reaction) in 50 mM phosphate buffer pH 7 at 50 °C for 30 minutes. The absorbance was measured at 540 nm. The measured values were background corrected. W168: Spore of B. subtilis W168. BsEglS-L1, BsEglS-L2, BsEglS-L3: Spores of B. subtilis W168 displaying BsEglS fused to CotY spore coat protein via linker L1, L2 and L3, respectively. The assay was performed in two biological replicates (N = 2).

Determination of optimal temperature

To characterize the enzymes immobilized on spores that exhibit endoglucanase activity (BsEglS-L1 ( BBa_K5117038), BsEglS-L2 ( BBa_K5117039) and BsEglS-L3 ( BBa_K5117037)), their optimum temperature was determined. A DNS assay was carried out for 30 minutes with CMC as the substrate at varying temperatures ranging from 40 °C to 90 °C (see Fig. 9). The assay was conducted according to the standard procedure, with the incubation temperature varied from 40 °C to 90 °C (see Experimentspage).

The highest activity was observed at 60 °C, and all activity values were normalized to this value. In general, the activity profiles were similar for all linker variants, with BsEglS-L1 showing the highest activity values at all temperatures tested. Earlier, Aa et al. (1994) demonstrated that the maximum activity of BsEglS is reached at 65 °C, which is consistent with the results obtained using spore solutions in this study. Therefore, the immobilization of the enzyme did not have a notable influence on the optimal temperature.

Fig. 9: Determination of temperature optimum of BsEglS displaying spores (BsEglS-L1 ( BBa_K5117038), BsEglS-L2 ( BBa_K5117039) and BsEglS-L3 ( BBa_K5117037) by using DNS assay (see Experimentspage). DNS assay was performed in 50 mM phosphate buffer pH 7 at different temperatures varying from 40 °C to 90 °C. The absorbance was measured at 540 nm. The measured values were background corrected and normalized to the highest value obtained from the reaction which was performed at 60 °C with BsEglS-L1. W168: Spore of B. subtilis W168. BsEglS-L1, BsEglS-L2, BsEglS-L3: Spores of B. subtilis W168 displaying BsEglS fused to CotY spore coat protein via linker L1, L2 and L3, respectively.The amount of spores was adjusted to achieve an OD₆₀₀ of 0.2 in the reaction mixture. The assay was performed with one biological replicate (N = 1). Absorbance from the control was subtracted from the measured values to account for background signal.

Assessment of the thermostability of spore-displayed endoglucanases

Furthermore, enzyme thermostability was evaluated. Spore-containing solutions were incubated for 2 hours at different temperatures ranging from 40 °C to 90 °C and then cooled down to the room temperature prior to the assay (see Fig. 10). The measured absorbance values were background corrected and normalized to the corresponding absorbance values obtained with spore solutions stored at room temperature prior to the reaction.

It was shown that the enzymes lost their activity when incubated at temperatures higher than 60 °C. This result correlated with earlier findings of Aa et al. (1994). They could demonstrate that the enzyme retains its activity up to 55 °C but loses functionality when exposed to temperatures above this threshold for 30 minutes. Thus, the immobilization of endoglucanases did not improve the enzyme's thermostability.

Fig. 10: : Determination of thermostability of endoglucanase displaying spores (BsEglS-L1 ( BBa_K5117038), BsEglS-L2 ( BBa_K5117039) and BsEglS-L3 ( BBa_K5117037)) by using DNS assay. (see Experimentspage). DNS assay was performed with BsEglS displaying spores with final OD₆₀₀ of 0.2 in 50 mM phosphate buffer pH 7 at 50 °C for 30 minutes. Prior to reaction the spore solutions were incubated at different temperatures varying from 40 °C to 90 °C and then cooled down to room temperature. The absorbance was measured at 540 nm. The measured values were background corrected and normalized to the corresponding values obtained without the pre-incubation of the spore solution at higher temperatures. W168: Spore of B. subtilis W168. BsEglS-L1, BsEglS-L2, BsEglS-L3: Spores of B. subtilis W168 displaying BsEglS fused to CotY spore coat protein via linker L1, L2 and L3, respectively. The assay was performed with one biological replicate (N = 1).

In the future, the influence of longer preincubation times on enzyme activity should be studied to assess the potential efficiency of applying BsEglS at an industrial scale. Furthermore, all experiments performed with DNS assay should be repeated in triplicates to ensure more accurate enzyme activity determination. Additionally, methods for estimating the enzyme amount on the spore cells should be developed to enable better comparability of the results with values reported in the literature.

References

Aa K., Flengsrud R., Lindahl V., Tronsmo A. (1994): Characterization of production and enzyme properties of an endo-β-1,4-glucanase from Bacillus subtilis CK-2 isolated from compost soil. Antonie van Leeuwenhoek 66, 319–326.

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

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

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

Sharma, P., & Guptasarma, P. (2017). Endoglucanase activity at a second site in Pyrococcus furiosus triosephosphate isomerase – Promiscuity or compensation for a metabolic handicap? FEBS Open Bio, 7(8), 1126–1143. https://doi.org/10.1002/2211-5463.12249

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