Difference between revisions of "Part:BBa K5117044"

Latest revision as of 21:59, 2 December 2024

PcotYZ-BsRBS-BhrPET-L1-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)
gene of the uncultured bacterium HR29 encoding a polyethylene terephthalate hydrolase (PETase, EC 3.1.1.101),

a homologue of the leaf-branch compost cutinase (LCC), codon optimized for Bacillus subtilis (Xi et al. 2021) without any signal peptide,

addition of a short flexible linker (L1) downstream of the coding sequence encoding the amino acids GA (BBa_K5117032)

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 BhrPET on the spore crust of B. subtilis (spore surface display)

Potential application: Degradation of PET

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 1009
Illegal AgeI site found at 695
Illegal AgeI site found at 740
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_K5117032) 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studies, this promoter has so far provided the highest activity for spore surface display (Bartels et al. 2018, unpublished data of Elif Öztel).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 short flexible linker GA.

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-BhrPET generated in the subcloning phase served as template for PCR of the enzyme candidate (see BBa_K5117020). 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 BhrPET-L1. Oligonucleotides for amplification can be found on the Experiments page. The correct PCR product has a size of 833 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 86.5 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, L1-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 (L1-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-BhrPET-L1-cotY-B0014. Oligonucleotides for amplification can be found on the Experiments page. The correct PCR product has a size of 1650 bp. Other weak 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 192.8 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: 31.1 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-BhrPET-L1-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-BhrPET-L1-cotY-B0014 was successfully constructed (DNA concentration: 203.7 ng/µl).

Three Images with Different Sizes

Fig. 4: Agarose gel electrophoresis: Insert amplification of P_cotYZ-BhrPET-L1-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 1711 bp. The negative control displayed no bands. 1 kb Plus DNA Ladder (NEB) served as marker (M). Colony 4 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-BhrPET-L1-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 p-nitrophenyl-acetate (pNPAc) assay to achieve a final OD₆₀₀ of 0.2 in the reaction. Further details are available on the Experimentspage.

pNPAc assay for determination of activity

The PETase activity of spores displaying BhrPET (BhrPET-L1 ( BBa_K5117044), BhrPET-L2 ( BBa_K5117045) and BhrPET-L3 ( BBa_K5117046)) was evaluated using an activity assay based on the method described by Xi et al. (2020). To demonstrate the functionality of the immobilized BhrPET, a pNPAc assay was conducted. In this assay, pNPAc is degraded to acetate and p-nitrophenol (pNP), which imparts a yellow color to the solution (Xi et al. 2021, Kademi et al. 2000). Notably, the reaction also occurs in the absence of enzymes, which is referred to as autohydrolysis. To rule out the influence of autohydrolysis on the measured activity values, a negative control without spore solution was included.

The reaction mixture contained phosphate buffer, spores with a final OD₆₀₀ of 0.2, pNPAc at a final concentration of 1 mM, and deionized water, and was incubated at 40 °C for 10 minutes. Detailed protocol can be found on the Experimentspage. Following incubation, the reaction was centrifuged at 13,000 rpm for 1 minute to avoid the spores interfering with the absorbance measurement. After centrifugation, 100 µl of the supernatant was transferred into a 96-well plate for absorbance measurement at 405 nm, performed in triplicates.

The PETase activity assay results for spores displaying BhrPET with different linkers are summarized in Fig. 6. The controls included reactions without spore solution and with W168 spores, which did not display enzymes, serving as negative controls. The absorbance values at 405 nm, with the control subtracted, indicate the enzyme activity. W168 control showed negligible absorbance, confirming the absence of enzymatic activity. Among the variants, BhrPET-L3 exhibited the highest activity, with an absorbance of approximately 0.9, followed closely by BhrPET-L1 at around 0.8. BhrPET-L2 showed a lower activity compared to the other linker variants, with an absorbance of about 0.5. These results suggest that the choice of linkers has a substantial effect on the enzyme's performance, with BhrPET-L3 being the most effective under the tested conditions.

Fig. 6: Activity assay of BhrPET-displaying spores ((BhrPET-L1 ( BBa_K5117044), BhrPET-L2 ( BBa_K5117045) and BhrPET-L3 ( BBa_K5117046)) after 10 minutes of incubation at 40 °C with pNPAc as the substrate (see Experimentspage). Absorbance was measured at 405 nm, indicating the formation of pNP, using 100 µl of the reaction supernatant in the reaction mixture. Measurements were performed in triplicates (n = 3), and mean values were calculated. Controls included W168 spores, which did not display any enzyme, and a reaction without spores, which served as a blank. The data represent absorbance values with the control values subtracted, illustrating the enzyme activity for each construct. The amount of spores was adjusted to achieve an OD₆₀₀ of 0.2 in the reaction mixture. The assay was performed in one biological replicates (N = 1). The absorbance from this control was subtracted from the measured values to account for background signal.

The initial PETase activity assay showed fluctuations in activity between the constructs, which may have been influenced by the very low yield of spores in the experiment. To address this issue, the spore preparation was repeated twice for subsequent tests to ensure consistent results. The procedure was repeated as previously described. Unfortunately, the yield of BhrPET-L2 spores was too low to perform any experiments. The Fig. 7 displays the mean absorbance values of two replicates, with the control (substrate without spores) subtracted. The results indicate that BhrPET-L1 exhibited the highest enzyme activity, with a maximum absorbance of approximately 2.5, while BhrPET-L3 showed slightly lower activity. Due to the higher performance, all subsequent experiments testing thermostability and optimal temperature were conducted using BhrPET-L1, as its spore yield was also the highest (data not shown).

Additionally, after the spore preparation, the spores appeared impure, which suggests there may have been issues with the sporulation of the PET constructs. This means that the actual enzyme activity might be higher than what was observed.

Fig. 7: Activity assay of BhrPET-displaying spores ((BhrPET-L1 ( BBa_K5117044) and BhrPET-L3 ( BBa_K5117046)) after 10 minutes of incubation at 40 °C with pNPAc as the substrate (see Experimentspage). Absorbance was measured at 405 nm, indicating the formation of pNP, using 100 µl of the reaction supernatant in the reaction mixture. Measurements were performed in triplicates (n = 3), and mean values were calculated. Controls included W168 spores, which did not display any enzyme, and a reaction without spores, which served as a blank. The data represent absorbance values with the control values subtracted, illustrating the enzyme activity for each construct. 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.

Determination of optimal temperature

The optimal temperature for BhrPET-L1 was determined by conducting the enzyme activity assay at varying temperatures ranging from 40 °C to 90 °C for 10 minutes. The results of the temperature-dependent activity assay for BhrPET-L1 are shown in Fig. 8. The graph illustrates the relative absorbance at 405 nm for W168 and BhrPET-L1 spores, with values normalized to the highest observed absorbance (A₄₀₅ = 2.35) for BhrPET-L1 at 50 °C, set as 100 %. The W168 control demonstrated negligible absorbance across all temperatures, confirming the absence of enzymatic activity. BhrPET-L1 showed nearly 90% of its maximum activity at 40 °C and around 80% at 60 °C. However, at 70 °C, enzyme activity sharply declined to approximately 10%, and no activity was detected at 80 °C or 90 °C. These findings suggest that the optimal temperature range for BhrPET-L1 is between 40 °C and 50 °C. Temperatures above 60 °C impair the enzyme's stability and catalytic performance, likely due to the thermal instability of either the enzyme or the substrate.

Fig. 8: Relative activity of BhrPET-L1 and W168 spores at varying temperatures (40 °C to 90 °C) for determining the optimal temperature for enzyme activity with pNPAc as the substrate (see Experimentspage). The x-axis represents temperature (°C). The y-axis shows relative activity as a percentage, normalized to the highest observed value at 50 °C (100%, A₄₀₅ = 2.35). The amount of spores was adjusted to achieve an OD₆₀₀ of 0.2 in the reaction mixture. Absorbance was measured at 405 nm, indicating the formation of pNP, using 100 µl of the reaction supernatant in the reaction mixture. Measurements were performed in triplicates (n = 3), and mean values were calculated. BhrPET-L1 demonstrated peak activity at 50 °C, with about 90% activity remaining at 40 °C and 80% at 60 °C. A reduction in activity was noted at 70 °C, dropping to 10%, and no enzyme activity was detectable at 80 °C or 90 °C. The assay was performed in one biological replicate (N = 1).

Assessment of the thermostability of spore-displayed PETase

Spore solutions were incubated at various temperatures ranging from 40 °C to 90 °C for 2 hours to evaluate the thermostability of the BhrPET-L1 displayed on the spores. After heat treatment, the samples were allowed to cool to room temperature. Subsequently, the residual enzyme activity of BhrPET-L2 was determined using an assay previously described (for 10 minutes at 40 °C). The results of this assessment are presented in Fig. 9.

The relative activity of BhrPET-L1 and W168 spores were evaluated after preincubation at various temperatures ranging from 40 °C to 90 °C for 2 hours. The measured values were background corrected and normalized to the corresponding values obtained without the pre-incubation of the spore solution at higher temperatures.

BhrPET-L1 retained substantial activity up to 60 °C, indicating good thermostability under these conditions. However, enzyme activity declined above 60 °C, with minimal activity detected at 70 °C (20 %) and 80 °C (approx. 17%), and almost no activity observed at 90 °C. This indicates that BhrPET-L1 loses both stability and catalytic function at higher temperatures. The W168 control showed negligible activity across all temperatures, confirming that the observed enzymatic activity in BhrPET-L1 is specific to the enzyme displayed on the spores.

BhrPET-L1 demonstrates promising activity within the 40 °C to 50 °C temperature range and maintains good thermostability up to 60 °C, making it suitable for applications within moderate temperature environments. However, the enzyme's stability and activity diminish at temperatures above 60 °C.

Noteworthy, the obtained results contradict earlier findings from the work of Xi et al (2020), in which almost a linear increase of activity was observed from 30 °C to 90 °C after assessment of the optimal temperature. In addition, with the thermostability experiments performed by Xi et al (2020) it was shown that BhrPET could retain 80 % of its activity when pre-incubated at 80 °C for 2 hours, whereas in our project a loss of BhrPET activity was detected after its pre-incubation for 2 hours at 70 °C and higher temperatures. The discrepancies in the results could be influenced by multiple factors. First, Xi et al (2020) applied different assay conditions and used p-nitrophenyl-octanoate and not pNPAc as a substrate. Second, the solutions with spore displaying BhrPET contained some impurities as described above, which could have interfered with the applied assay. Finally, enzyme immobilization on the spore surface could resulted in a negative impact on the protein activity and stability.

Fig. 9: Relative activity of BhrPET-L1 and W168 spores for determining the thermostability of the enzymes displayed on the spores, which were preincubated at varying temperatures (40 °C to 90 °C) for 2 hours using pNPAc assay (see Experimentspage). Following the pre-incubation, the reaction with pNPAc was conducted for 10 minutes at 40 °C, and the formation of pNP was measured by absorbance at 405 nm. Spores from the W168 strain were used as a control, along with additional control without spore solution. The amount of spores was adjusted to achieve an OD₆₀₀ of 0.2 in the reaction mixture. The absorbance from the control without spores was subtracted from the measured values to account for background signal. The measured values were background corrected and normalized to the corresponding values obtained without the pre-incubation of the spore solution at higher temperatures. The experiment was performed in one biological replicate (N = 1). Measurements were performed in duplicates, and mean values were calculated.

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

Kademi, A., Ait-Abdelkader, N., Fakhreddine, L., & Baratti, J. (2000). Purification and characterization of a thermostable esterase from the moderate thermophile Bacillus circulans. Applied Microbiology and Biotechnology, 54(2), 173–179. https://doi.org/10.1007/S002530000353

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

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

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

Xi, X., Ni, K., Hao, H., Shang, Y., Zhao, B., & Qian, Z. (2021). Secretory expression in Bacillus subtilis and biochemical characterization of a highly thermostable polyethylene terephthalate hydrolase from bacterium HR29. Enzyme and Microbial Technology, 143, 109715. https://doi.org/10.1016/J.ENZMICTEC.2020.109715

@@ Line 10: / Line 10: @@
 <li>ribosome binding site of <i>Bacillus subtilis</i> <html><a href="https://parts.igem.org/Part:BBa_K5117000">(BBa_K5117000)</a></html></li>
-- gene of the uncultured bacterium HR29 encoding a polyethylene terephthalate hydrolase (PETase, EC 3.1.1.101), a homologue of the leaf-branch compost cutinase (LCC), codon optimized for <i>Bacillus subtilis</i> (Xi <i>et al.</i> 2021) without any signal peptide,
+<li>gene of the uncultured bacterium HR29 encoding a polyethylene terephthalate hydrolase (PETase, EC 3.1.1.101), </li>
-<div style="text-indent: 1px;">addition of a short flexible linker (L1) downstream of the coding sequence encoding the amino acids GA <html><a href="https://parts.igem.org/Part:BBa_K5117032"(BBa_K5117032)</a></html></div>
+<div style="text-indent: 1px;">a homologue of the leaf-branch compost cutinase (LCC), codon optimized for <i>Bacillus subtilis</i> (Xi <i>et al.</i> 2021) without any signal peptide,</div>
+<div style="text-indent: 1px;">addition of a short flexible linker (L1) downstream of the coding sequence encoding the amino acids GA <html><a href="https://parts.igem.org/Part:BBa_K5117032">(BBa_K5117032)</a></html></div>
 <li><i>cotY</i> gene of <i>Bacillus subtilis</i> <html><a href="https://parts.igem.org/Part:BBa_K5117022">(BBa_K5117022)</a></html></li>
@@ Line 26: / Line 28: @@
 <b>Main purpose of use:</b> Immobilization of BhrPET on the spore crust of <i>B. subtilis</i> (spore surface display)
-<b>Application:</b> Degradation of PET
+<b>Potential application:</b> Degradation of PET
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K5117044 SequenceAndFeatures</partinfo>
+===Enzyme characterization according to literature===
+<p> The characterization of the enzyme included in this composite part can be found on the basic part page <html><a href="https://parts.igem.org/Part:BBa_K5117032">(BBa_K5117032)</a></html> of the enzyme. </p>
+===Construct design===
+For compatibility with the BioBrick RFC[10] standard, the restriction sites <i>Eco</i>RI, <i>Xba</i>I, <i>Spe</i>I, <i>Pst</i>I and <i>Not</i>I were removed from the coding sequence (CDS). To make the part compatible with the Type IIS standard, <i>Bsa</i>I and <i>Sap</i>I sites were removed as well. This was achieved by codon exchange using the codon usage table of <i>Bacillus subtilis</i> <html><a href="https://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=1423&aa=1&style=N">(Codon Usage Database Kazusa)</a></html>.
+To express target genes only under sporulation, the sporulation-dependent promoter P<i><sub>cotYZ</sub></i> of <i>B. subtilis</i> was chosen. In previous studies, this promoter has so far provided the highest activity for spore surface display (Bartels <i>et al.</i> 2018, unpublished data of Elif Öztel). The promoter was followed by the ribosome binding site (RBS) for the host <i>B. subtilis</i> 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 <i>et al.</i> 2013, Bartels <i>et al.</i> 2018, Lin <i>et al.</i> 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 <i>et al.</i> 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)<sub>4</sub> (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 <i>et al.</i> 2013).
+The composite part documented in this page contains the short flexible linker GA.
+Following the CDS of <i>cotY</i>, a spacer consisting of 10 bp of the natural genome sequence downstream from the <i>cotYZ</i> 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 <html><a href="https://parts.igem.org/Part:BBa_J179000">pBS1C</a></html> from the <i>Bacillus</i> BioBrickBox was used as an integrative plasmid backbone enabling genomic integration into the <i>amyE</i> locus of <i>B. subtilis</i> (Radeck <i>et al.</i> 2013).
+===Construction of spore display plasmids===
+First, all biological parts including the enzyme candidate (Fig. 1) as well as the promoter P<sub><i>cotYZ</i></sub>, the terminator B0014 and the anchor gene <i>cotY</i> (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-BhrPET generated in the subcloning phase served as template for PCR of the enzyme candidate (see <html><a href="https://parts.igem.org/Part:BBa_K5117020">BBa_K5117020</a></html>). The linker was added by oligonucleotides. The promoter and anchor gene were amplified from genomic DNA of <i>B. subtilis</i> W168 and the terminator from a plasmid provided by the laboratory collection of Prof. Thorsten Mascher (General Microbiology, TU Dresden).
+<html><center><img src="https://static.igem.wiki/teams/5117/parts-registry/spore-display/bhrpet/bhrpet-l1-l2-l3-part.png" style="width: 40%; height: auto;"></center></html>
+<p class="image_caption"><center> <font size="1"><b> Fig. 1: Agarose gel electrophoresis: PCR of BhrPET-L1. </b> Oligonucleotides for amplification can be found on the <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html> page. The correct PCR product has a size of 833 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 86.5 ng/µl. </font></center></p>
+<html><center><img src="https://static.igem.wiki/teams/5117/parts-registry/spore-display/spore-parts.png" style="width: 75%; height: auto;"></center></html>
+<p class="image_caption"><center> <font size="1"><b> Fig. 2: Agarose gel electrophoresis: PCR of P<sub><i>cotYZ</i></sub>, <i>cotY</i> and B0014. </b> Oligonucleotides for amplification can be found on the <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html> page. Amplification of P<sub><i>cotYZ</i></sub> results in a band of 238 bp, L1-<i>cotY</i> 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<sub><i>cotYZ</i></sub>), 99.4 ng/µl (L1-<i>cotY</i>), and 52.2 ng/µl (B0014). </font></center></p>
+The composite part was subsequently assembled via Overlap PCR by complementary overhangs, which were designed and added by oligonucleotides (Fig. 3).
+<html><center><img src="https://static.igem.wiki/teams/5117/parts-registry/spore-display/bhrpet/bhrpet-l1-ov.png
+" style="width: 30%; height: auto;"></center></html>
+<p class="image_caption"><center> <font size="1"><b> Fig. 3: Agarose gel electrophoresis: Overlap PCR of the spore display construct P<sub><i>cotYZ</i></sub>-BhrPET-L1-<i>cotY</i>-B0014. </b> Oligonucleotides for amplification can be found on the <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html> page. The correct PCR product has a size of 1650 bp. Other weak 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 192.8 ng/µl. </font></center></p>
+Afterwards, the Overlap PCR product was cloned into the vector backbone pBS1C enabling genome integration into the <i>amyE</i> locus in <i>B. subtilis</i> (Radeck <i>et al.</i> 2013). For that purpose, the insert as well as pBS1C were digested with <i>Eco</i>RI and <i>Pst</i>I and purified by PCR clean up (DNA concentration: 31.1 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 <i>E. coli</i> 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<sub><i>cotYZ</i></sub>-BhrPET-L1-<i>cotY</i>-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<sub><i>cotYZ</i></sub>-BhrPET-L1-<i>cotY</i>-B0014 was successfully constructed (DNA concentration: 203.7 ng/µl).
+<html lang="en">
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+    <title>Three Images with Different Sizes</title>
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+</head>
+<body>
+<div class="clearfix">
+    <div class="image-container image1">
+        <img src="https://static.igem.wiki/teams/5117/parts-registry/spore-display/bhrpet/bhrpet-l1-ecoli.png" alt="Image 1">
+    </div>
+    <div class="image-container image2">
+        <img src="https://static.igem.wiki/teams/5117/parts-registry/spore-display/bhrpet/bhrpet-spore-ecoli-control.png" alt="Image 2">
+    </div>
+</div>
+</body>
+</html>
+<p class="image_caption"><center> <font size="1"><b> Fig. 4: Agarose gel electrophoresis: Insert amplification of P<sub><i>cotYZ</i></sub>-BhrPET-L1-<i>cotY</i>-B0014 by Colony PCR of transformed <i>E. coli</i> DH10β cells. </b>
+ Oligonucleotides for amplification can be found on the <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html> page. Numbers 1-4 correspond to chosen colonies. The correct PCR product has a size of 1711 bp. The negative control displayed no bands. 1 kb Plus DNA Ladder (NEB) served as marker (M). Colony 4 was verified by sequencing and contained the correct insert sequence. </font></center></p>
+===Generation of <i>Bacillus subtilis</i> strains===
+Ultimately, this plasmid was transformed into the <i>B. subtilis</i> 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<sub><i>cotYZ</i></sub>-BhrPET-L1-<i>cotY</i>-B0014, which was linearized by restriction with <i>Bsa</i>I 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 <i>amyE</i> locus was successful, the native amylase of <i>Bacillus</i> 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 <i>B. subtilis</i> strain were chosen for cryo-conservation, with clone 1 being used for subsequent activity tests.
+<html><center><img src="https://static.igem.wiki/teams/5117/parts-registry/spore-display/bhrpet/bhrpet-l1-starch-assay.png" style="width: 50%; height: auto;"></center></html>
+<p class="image_caption"><center> <font size="1"><b> Fig. 5: Starch assay of transformed <i>B. subtilis</i> W168 cells. </b>
+ 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. </font></center></p>
+===Spore preparation===
+Spores were prepared by culturing cells in LB medium with chloramphenicol until they reached the exponential growth phase (OD<sub>600</sub> 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 dH<sub>2</sub>O and SDS to remove vegetative cell residues. The spore suspension was adjusted to an OD<sub>600</sub> of 2 for the <i>p</i>-nitrophenyl-acetate (pNPAc) assay to achieve a final OD<sub>600</sub> of 0.2 in the reaction. Further details are available on the <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html>page.
+===pNPAc assay for determination of activity===
+The PETase activity of spores displaying BhrPET (BhrPET-L1 (<html><a href="https://parts.igem.org/Part:BBa_K5117044"> BBa_K5117044</a></html>), BhrPET-L2 (<html><a href="https://parts.igem.org/Part:BBa_K5117045"> BBa_K5117045</a></html>) and BhrPET-L3 (<html><a href="https://parts.igem.org/Part:BBa_K5117046"> BBa_K5117046</a></html>)) was evaluated using an activity assay based on the method described by <i>Xi et al.</i> (2020). To demonstrate the functionality of the immobilized BhrPET, a pNPAc assay was conducted. In this assay, pNPAc is degraded to acetate and <i>p</i>-nitrophenol (pNP), which imparts a yellow color to the solution (Xi <i>et al. </i> 2021, Kademi <i>et al. </i> 2000). Notably, the reaction also occurs in the absence of enzymes, which is referred to as autohydrolysis. To rule out the influence of autohydrolysis on the measured activity values, a negative control without spore solution was included.
+The reaction mixture contained phosphate buffer, spores with a final OD<sub>600</sub> of 0.2, pNPAc at a final concentration of 1 mM, and deionized water, and was incubated at 40 °C for 10 minutes. Detailed protocol can be found on the <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html>page. Following incubation, the reaction was centrifuged at 13,000 rpm for 1 minute to avoid the spores interfering with the absorbance measurement. After centrifugation, 100 µl of the supernatant was transferred into a 96-well plate for absorbance measurement at 405 nm, performed in triplicates.
+The PETase activity assay results for spores displaying BhrPET with different linkers are summarized in Fig. 6. The controls included reactions without spore solution and with W168 spores, which did not display enzymes, serving as negative controls. The absorbance values at 405 nm, with the control subtracted, indicate the enzyme activity. W168 control showed negligible absorbance, confirming the absence of enzymatic activity. Among the variants, BhrPET-L3 exhibited the highest activity, with an absorbance of approximately 0.9, followed closely by BhrPET-L1 at around 0.8. BhrPET-L2 showed a lower activity compared to the other linker variants, with an absorbance of about 0.5. These results suggest that the choice of linkers has a substantial effect on the enzyme's performance, with BhrPET-L3 being the most effective under the tested conditions.
+<html> <center><img src="https://static.igem.wiki/teams/5117/parts-registry/assays-spore-display/petase/bhrpet1.png" style="width: 50%; height: auto;"></center> </html><p class="image_caption"><center><font size="1"><b>Fig. 6: Activity assay of BhrPET-displaying spores ((BhrPET-L1 (<html><a href="https://parts.igem.org/Part:BBa_K5117044"> BBa_K5117044</a></html>), BhrPET-L2 (<html><a href="https://parts.igem.org/Part:BBa_K5117045"> BBa_K5117045</a></html>) and BhrPET-L3 (<html><a href="https://parts.igem.org/Part:BBa_K5117046"> BBa_K5117046</a></html>)) after 10 minutes of incubation at 40 °C with pNPAc as the substrate (see <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html>page).</b> Absorbance was measured at 405 nm, indicating the formation of pNP, using 100 µl of the reaction supernatant in the reaction mixture. Measurements were performed in triplicates (n = 3), and mean values were calculated. Controls included W168 spores, which did not display any enzyme, and a reaction without spores, which served as a blank. The data represent absorbance values with the control values subtracted, illustrating the enzyme activity for each construct. The amount of spores was adjusted to achieve an OD<sub>600</sub> of 0.2 in the reaction mixture. The assay was performed in one biological replicates (N = 1). The absorbance from this control was subtracted from the measured values to account for background signal. </font></center></p>
+The initial PETase activity assay showed fluctuations in activity between the constructs, which may have been influenced by the very low yield of spores in the experiment. To address this issue, the spore preparation was repeated twice for subsequent tests to ensure consistent results. The procedure was repeated as previously described. Unfortunately, the yield of BhrPET-L2 spores was too low to perform any experiments. The Fig. 7 displays the mean absorbance values of two replicates, with the control (substrate without spores) subtracted. The results indicate that BhrPET-L1 exhibited the highest enzyme activity, with a maximum absorbance of approximately 2.5, while BhrPET-L3 showed slightly lower activity. Due to the higher performance, all subsequent experiments testing thermostability and optimal temperature were conducted using BhrPET-L1, as its spore yield was also the highest (data not shown).
+Additionally, after the spore preparation, the spores appeared impure, which suggests there may have been issues with the sporulation of the PET constructs. This means that the actual enzyme activity might be higher than what was observed.
+<html> <center><img src="https://static.igem.wiki/teams/5117/parts-registry/assays-spore-display/petase/bhrpet2.png" style="width: 50%; height: auto;"></center> </html><p class="image_caption"><center><font size="1"><b>Fig. 7: Activity assay of BhrPET-displaying spores ((BhrPET-L1 (<html><a href="https://parts.igem.org/Part:BBa_K5117044"> BBa_K5117044</a></html>) and BhrPET-L3 (<html><a href="https://parts.igem.org/Part:BBa_K5117046"> BBa_K5117046</a></html>)) after 10 minutes of incubation at 40 °C with pNPAc as the substrate (see <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html>page). </b> Absorbance was measured at 405 nm, indicating the formation of pNP, using 100 µl of the reaction supernatant in the reaction mixture. Measurements were performed in triplicates (n = 3), and mean values were calculated. Controls included W168 spores, which did not display any enzyme, and a reaction without spores, which served as a blank. The data represent absorbance values with the control values subtracted, illustrating the enzyme activity for each construct. The amount of spores was adjusted to achieve an OD<sub>600</sub> 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. </font></center></p>
+===Determination of optimal temperature===
+The optimal temperature for BhrPET-L1 was determined by conducting the enzyme activity assay at varying temperatures ranging from 40 °C to 90 °C for 10 minutes. The results of the temperature-dependent activity assay for BhrPET-L1 are shown in Fig. 8.
+The graph illustrates the relative absorbance at 405 nm for W168 and BhrPET-L1 spores, with values normalized to the highest observed absorbance (A<sub>405</sub> = 2.35) for BhrPET-L1 at 50 °C, set as 100 %. The W168 control demonstrated negligible absorbance across all temperatures, confirming the absence of enzymatic activity. BhrPET-L1 showed nearly 90% of its maximum activity at 40 °C and around 80% at 60 °C. However, at 70 °C, enzyme activity sharply declined to approximately 10%, and no activity was detected at 80 °C or 90 °C. These findings suggest that the optimal temperature range for BhrPET-L1 is between 40 °C and 50 °C. Temperatures above 60 °C impair the enzyme's stability and catalytic performance, likely due to the thermal instability of either the enzyme or the substrate.
+<html> <center><img src="https://static.igem.wiki/teams/5117/parts-registry/assays-spore-display/petase/bhrpet-opt-t.png" style="width: 50%; height: auto;"></center> </html><p class="image_caption"><center><font size="1"><b>Fig. 8: Relative activity of BhrPET-L1 and W168 spores at varying temperatures (40 °C to 90 °C) for determining the optimal temperature for enzyme activity with pNPAc as the substrate (see <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html>page). </b> The x-axis represents temperature (°C). The y-axis shows relative activity as a percentage, normalized to the highest observed value at 50 °C (100%, A<sub>405</sub> = 2.35). The amount of spores was adjusted to achieve an OD<sub>600</sub> of 0.2 in the reaction mixture. Absorbance was measured at 405 nm, indicating the formation of pNP, using 100 µl of the reaction supernatant in the reaction mixture. Measurements were performed in triplicates (n = 3), and mean values were calculated. BhrPET-L1 demonstrated peak activity at 50 °C, with about 90% activity remaining at 40 °C and 80% at 60 °C. A reduction in activity was noted at 70 °C, dropping to 10%, and no enzyme activity was detectable at 80 °C or 90 °C. The assay was performed in one biological replicate (N = 1). </font></center></p>
+===Assessment of the thermostability of spore-displayed PETase===
+Spore solutions were incubated at various temperatures ranging from 40 °C to 90 °C for 2 hours to evaluate the thermostability of the BhrPET-L1 displayed on the spores. After heat treatment, the samples were allowed to cool to room temperature. Subsequently, the residual enzyme activity of BhrPET-L2 was determined using an assay previously described (for 10 minutes at 40 °C). The results of this assessment are presented in Fig. 9.
+The relative activity of BhrPET-L1 and W168 spores were evaluated after preincubation at various temperatures ranging from 40 °C to 90 °C for 2 hours. The measured values were background corrected and normalized to the corresponding values obtained without the pre-incubation of the spore solution at higher temperatures.
+BhrPET-L1 retained substantial activity up to 60 °C, indicating good thermostability under these conditions. However, enzyme activity declined above 60 °C, with minimal activity detected at 70 °C (20 %) and 80 °C (approx. 17%), and almost no activity observed at 90 °C. This indicates that BhrPET-L1 loses both stability and catalytic function at higher temperatures. The W168 control showed negligible activity across all temperatures, confirming that the observed enzymatic activity in BhrPET-L1 is specific to the enzyme displayed on the spores.
+BhrPET-L1 demonstrates promising activity within the 40 °C to 50 °C temperature range and maintains good thermostability up to 60 °C, making it suitable for applications within moderate temperature environments. However, the enzyme's stability and activity diminish at temperatures above 60 °C.
+Noteworthy, the obtained results contradict earlier findings from the work of Xi <i>et al</i> (2020), in which almost a linear increase of activity was observed from 30 °C to 90 °C after assessment of the optimal temperature. In addition, with the thermostability experiments performed by Xi <i>et al</i> (2020) it was shown that BhrPET could retain 80 % of its activity when pre-incubated at 80 °C for 2 hours, whereas in our project a loss of BhrPET activity was detected after its pre-incubation for 2 hours at 70 °C and higher temperatures. The discrepancies in the results could be influenced by multiple factors. First, Xi <i>et al</i> (2020) applied different assay conditions and used p-nitrophenyl-octanoate and not pNPAc as a substrate. Second, the solutions with spore displaying BhrPET contained some impurities as described above, which could have interfered with the applied assay. Finally, enzyme immobilization on the spore surface could resulted in a negative impact on the protein activity and stability.
+<html> <center><img src="https://static.igem.wiki/teams/5117/parts-registry/assays-spore-display/petase/bhrpet-thermostability.png" style="width: 50%; height: auto;"></center> </html><p class="image_caption"><center><font size="1"><b>Fig. 9: Relative activity of BhrPET-L1 and W168 spores for determining the thermostability of the enzymes displayed on the spores, which were preincubated at varying temperatures (40 °C to 90 °C) for 2 hours using pNPAc assay (see <html><a href="https://2024.igem.wiki/tu-dresden/experiments">Experiments</a></html>page). </b> Following the pre-incubation, the reaction with pNPAc was conducted for 10 minutes at 40 °C, and the formation of pNP was measured by absorbance at 405 nm. Spores from the W168 strain were used as a control, along with additional control without spore solution. The amount of spores was adjusted to achieve an OD<sub>600</sub>  of 0.2 in the reaction mixture. The absorbance from the control without spores was subtracted from the measured values to account for background signal. The measured values were background corrected and normalized to the corresponding values obtained without the pre-incubation of the spore solution at higher temperatures. The experiment was performed in one biological replicate (N = 1). Measurements were performed in duplicates, and mean values were calculated. </font></center></p>
+===References===
+Bartels J., López Castellanos S., Radeck J., Mascher T. (2018): Sporobeads: The utilization of the <i>Bacillus subtilis</i> 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
+Kademi, A., Ait-Abdelkader, N., Fakhreddine, L., & Baratti, J. (2000). Purification and characterization of a thermostable esterase from the moderate thermophile <i> Bacillus circulans</i>. Applied Microbiology and Biotechnology, 54(2), 173–179. https://doi.org/10.1007/S002530000353
+Lin P., Yuan H., Du J., Liu K., Liu H., Wang T. (2020): Progress in research and application development of surface display technology using <i>Bacillus subtilis</i> spores. Applied microbiology and biotechnology 104 (6), 2319–2331. https://doi.org/10.1007/s00253-020-10348-x
+McKenney P. T., Driks A., Eichenberger P. (2013): The <i>Bacillus subtilis</i> endospore: assembly and functions of the multilayered coat. Nature Reviews Microbiology 11 (1), 33–44. https://doi.org/10.1038/nrmicro2921
+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 <i>Bacillus</i> BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with <i>Bacillus subtilis</i>. 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 <i>Bacillus subtilis</i> and <i>Escherichia coli in vivo</i>. Molecular microbiology 6(9), 1105-1114. https://doi.org/10.1111/j.1365-2958.1992.tb01548.x
+Xi, X., Ni, K., Hao, H., Shang, Y., Zhao, B., & Qian, Z. (2021). Secretory expression in <i> Bacillus subtilis</i> and biochemical characterization of a highly thermostable polyethylene terephthalate hydrolase from bacterium HR29. Enzyme and Microbial Technology, 143, 109715. https://doi.org/10.1016/J.ENZMICTEC.2020.109715