iGEM 2024 — Team tu-dresden

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+ .
+ OUTLINE
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- [{{content.principles\_title}}](#href_key)
- [{{content.engineering\_title}}](#href_cycle)

[Engineering in Our Project
![](https://static.igem.wiki/teams/5117/images-wiki/plus-circle.svg)](#submenu2)

- [{{content.design\_title}}](#href_design)
- [{{content.build\_title}}](#href_build)
- [{{content.test\_title}}](#href_test)
- [{{content.learn\_title}}](#href_learn)

+ [References](#href_references)

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# **{{content.intro\_title}}**

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## {{content.synthetic\_title}}

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![](https://static.igem.wiki/teams/5117/images-wiki/engineering/1-wordcloud-neu.jpg)

Figure 1: Word cloud of Synthetic Biology.

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![](https://static.igem.wiki/teams/5117/images-wiki/engineering/2-engineeringcycle.png)

Figure 2: Engineering cycle including four phases: design, build, test,
learn.

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![](https://static.igem.wiki/teams/5117/images-wiki/engineering/3-engineeringcycle.png)

Fig. 3: Engineering cycle of our project, including the main cycle in the
middle representing our entire project, and small neighboring gearwheels representing different aspects of our
project.

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#### **{{content.literature\_title}}**

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We defined following criteria:

* biosafety level: S1 Bacillota (for cellulases)
* gene size: < 2000 bp (max. 2500 bp)
* active state: monomer
* localization: extracellular (if possible)
* pH range: ≈ 5 - 8
* temperature: ≈ 30 - 60 °C

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Table 1: Enzyme candidates for cellulose and PET degradation.

| Enzyme | Candidate | Species | Phylum, Class | Abbreviation | Gene size [bp] | Protein size [kDa] | Localization | References |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Endoglucanase (endo-1,4-β-D-glucanase,   EC 3.2.1.4) | EglS | *Bacillus subtilis* | *Bacillota, Bacilli* | BsEglS | 1500 | 55 | extracellular | MacKay *et al.* (1986), Aa *et al.*(1994), Lindahl *et al.* (1994) UniProtKB: P10475 |
|  | EglA | *Bacillus pumilus* | *Bacillota, Bacilli* | BpEglA | 1980 | 73 | extracellular | Lima *et al.* (2005) UniProtKB: Q5YLG1 |
|  | CelA | *Acetivibrio thermocellus* | *Bacillota, Clostridia* | AtCelA | 1434 | 52-60 | extracellular | Béguin *et al.* (1985), Schmidt *et al.* (2001 and 2002), van der Veen *et al.*(2013), Kitago *et al.* (2006,2007,2011), Weng *et al.* (2022) UniProtKB: A3DC29 |
|  | CelG | *Acetivibrio thermocellus* | *Bacillota, Clostridia* | AtCelG | 1701 | 63 | extracellular | Lemaire and Béguin (1993),   UniProtKB: Q05332 |
| Exoglucanase (1,4-β-cellobiosidase,   EC 3.2.1.176) | CelO | *Acetivibrio thermocellus* | *Bacillota,Clostridia* | AtCelO | 1983 | 75 | unknown | Zverlov *et al.* (2002), Ahmad *et al.* (2023), UniProtKB: Q9L3J2 |
|  | CelS | *Acetivibrio thermocellus* | *Bacillota, Clostridia* | AtCelS | 2226 | 75-82 | extracellular | Wang *et al.* (1993),   Wang and Wu (1993), Kruus *et al.* (1995), Guimarães *et al.* (2002), Wilson *et al.* (2013), UniProtKB: A3DH67 |
| β-Glucosidase (1,4-β-glucosidase,   EC 3.2.1.21) | BglA | *Bacillus halodurans* | *Bacillota, Bacilli* | BhBglA | 1344 | 52 | intracellular | Naz *et al.* (2010), Takami *et al.* (2000), UniProtKB: Q9KBK3 |
|  | BglB | *Paenibacillus polymyxa* | *Bacillota, Bacilli* | PpBglB | 1347 | 52 | intracellular | González-Candelas *et al.* (1990), Isorna *et al.* (2006 and 2007), Tsabitah *et al.* (2024), UniProtKB: P22505 |
|  | BglA | *Acetivibrio thermocellus* | *Bacillota, Clostridia* | AtBglA | 1347 | 51-53 | intracellular | Gräbnitz *et al.* (1991), Ahmed *et al.* (2019), UniProtKB: P26208 |
| PETase (Polyethylene terephthalate hydrolase,   EC 3.1.1.101) | BhrPETase | *Bacterium HR29* | *Chloroflexi* | BhrPET | 867 | 25 | extracellular | Kato *et al.* (2018), Wang and Wang (2021), Xi *et al.* (2021), Wang *et al.* (2024), UniProtKB: A0A2H5Z9R5 |

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![](https://static.igem.wiki/teams/5117/images-wiki/engineering/4-constructdesign-subcloning-bhbgla-new.png)

Figure 4: Design of biological parts, demonstrated with enzyme candidate
BhBglA.
Prefix/Suffix = BioBrick prefix/suffix. RBS = ribosome binding site. *EcoR*I, *Xba*I, *Spe*I,
*Pst*I, *Not*I = restriction
sites.

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![](https://static.igem.wiki/teams/5117/images-wiki/engineering/5-psb1c3.png)

Figure 5: Plasmid map of pSB1C3.
P*lac* = lactose inducible promoter and *rfp* = red fluorescent protein (original insert
serving as placeholder).
pMB1 = origin of replication for *E. coli (Ec).* *cat* = chloramphenicol acetyltransferase (chloramphenicol
resistance). *EcoR*I, *Xba*I, *Spe*I, *Pst*I, *Not*I = restriction sites.

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![](https://static.igem.wiki/teams/5117/images-wiki/engineering/6-pbs0e-xylr-pxyla.png)

Figure 6: Plasmid map of the replicative expression vector
pBS0E-*xylR*-P*xylA*.
*xylR* = xylose repressor. P*xylA* = xylose inducible promoter. *rfp* = red
fluorescent protein (placeholder). *bla* =
β-lactamase (ampicillin resistance in *E. coli*). *erm* = erythromycin resistance gene (MLS resistance
in *B.
subtilis*). ColE1 = origin of replication in *E. coli (Ec)*. ori1030 = origin of replication in *B.
subtilis (Bs)*.
*EcoRI*, *Xba*I, *Spe*I, *Pst*I, *Not*I, *Sap*I, *Bsa*I = restriction sites.

![](https://static.igem.wiki/teams/5117/images-wiki/engineering/7-pbs2e-xylr-pxyla.png)

Figure 7: Plasmid map of the integrative expression vector
pBS2E-*xylR*-P*xylA*.
*xylR* = xylose repressor. P*xylA* = xylose inducible promoter. *rfp* = red
fluorescent protein (placeholder). *bla* =
β-lactamase (ampicillin resistance in *E. coli*). *erm* = erythromycin resistance gene (MLS resistance
in *B.
subtilis*). ColE1 = origin of replication in *E. coli (Ec)*. *lacA’/’lacA = lacA* locus fragments
of the *B. subtilis*
genome. *EcoR*I, *Xba*I, *Spe*I, *Pst*I, *Not*I, *Sap*I, *Bsa*I = restriction
sites.

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![](https://static.igem.wiki/teams/5117/images-wiki/engineering/8-mrna-secondary-structures.png)

Figure 8: Predicted mRNA secondary structures of the terminator B0014 without (A) and with (B) 10 bp spacer
upstream (RNAfold WebServer).

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![](https://static.igem.wiki/teams/5117/images-wiki/engineering/engineering-fig-9-constructdesign-correction.webp)

Figure 9: Construct design for spore surface display, demonstrated with the
enzyme BhBglA fused to the N-terminus of CotY.

![](https://static.igem.wiki/teams/5117/images-wiki/engineering/10-pbs1c-new.png)

Figure 10: Plasmid map of the integrative vector pBS1C.
P*lac* = lactose inducible promoter and *rfp* = red fluorescent protein (original insert
serving as placeholder).
*bla* = β-lactamase (ampicillin resistance in *E. coli*). *cat* = chloramphenicol acetyl
acetyltransferase
(chloramphenicol resistance in *B. subtilis*). pMB1 = origin of replication for *E. coli (Ec)*.
*amyE’/’amyE = amyE*
locus fragments of the *B. subtilis* genome. *EcoR*I, *Xba*I, *Spe*I, *Pst*I,
*Not*I, *Bsa*I = restriction sites.

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![]( https://static.igem.wiki/teams/5117/images-wiki/engineering/fig11.png)

Figure 11: Determination of exoglucanases activity on
LB-Agar-Avicel plates containing 1% avicel.
After the incubation period for 24 hours, congo red staining and subsequent destaining with 1 M NaCl
was performed. Ac: Commercially available enzyme mix with exoglucanase activity (10 µl); NC:
negative control (10 µlwater).

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![]( https://static.igem.wiki/teams/5117/images-wiki/engineering/fig12.png)

Figure 12: Determination of exoglucanases activity on
LB-Agar-Avicel plates containing 1% avicel.
After the incubation period for 24 hours, congo red staining and subsequent destaining with 1 M
NaCl
was performed. Ac: Commercially available enzyme mix with exoglucanase activity (10 µl); NC:
negative control (10 µlwater).

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![]( https://static.igem.wiki/teams/5117/images-wiki/engineering/fig13.png)

Figure 13: Cellulose overlay assay for determination of exoglucanase
activity.
10 μl of Accellerase 1500 (Ac) mix was added to LB agar plates, dried out and then 0.2 % cellulose
overlay was poured onto the plate. The plates were incubated at 37 °C. After the incubation, the
plates were stained with congo red solution and subsequently destained with 1 M NaCl.

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![]( https://static.igem.wiki/teams/5117/images-wiki/engineering/fig14.png)

Figure 14: PASC and PASA assays for determination of exoglucanase
activity.
Arrow indicates zone of clearance which indicate exoglucanase activity.
As sample served 20 µl of commercially available cellobiohydrolase I.

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![]( https://static.igem.wiki/teams/5117/images-wiki/engineering/fig15.png)

Figure 15: LB-Agar-Esculin plates to determine β-glucosidase
activity of WB800N transformed with pBS0E-*xylR*-P*xylA* vector carrying
AtBglA, BhBglA or PpBglB (pBS0EX-AtBglA), pBS0EX-BhBglA, pBS0EX-PpBglB.
The cultures of both clones and the negative control (WB800N strain and *E. coli*) were grown
overnight. Next day 10 µl of overnight culture with OD600 of 0.5, was added to the
plate and then incubated for 24 hours at 37 °C. The plates demonstrate the formation of black
halos around all cultures, including the negative control, indicating esculin degradation and
suggesting high background activity from *B. subtilis* compared to *E. coli* (no
black halo formation). No increase in activity was observed on plates containing xylose as an
inducer.

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![]( https://static.igem.wiki/teams/5117/images-wiki/engineering/fig16.png)

Figure 16: Glucose concentration determination following
cellobiose degradation by intracellular β-glucosidases (lysate of pBS0EX-AtBglA, pBS0EX-BhBglA,
pBS0EX-PpBglB) by using the Amplex™ Red Glucose/Glucose Oxidase Assay Kit.
The absorbance was measured at 560 nm, representing glucose production after degradation of 50 mM
cellobiose by different β-glucosidases from lysate. WB800N was used as negative control. Dark
purple bars represent uninduced (control) samples, light purple bars represent induced (0.5 %
xylose) samples, and the green bar represents the 50 mM cellobiose standard.

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![]( https://static.igem.wiki/teams/5117/images-wiki/engineering/fig17.png)

Figure 17: Varying percentages of lysate were mixed with 50 mM
cellobiose, 200 µM glucose, and 1X reaction buffer from the Amplex™ Red Glucose/Glucose Oxidase
Assay Kit.
Glucose concentration was measured using the kit, and absorbance was recorded at 560 nm. The
absorbance values were normalized to the reaction without lysate.

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![]( https://static.igem.wiki/teams/5117/images-wiki/engineering/fig18.png)

Figure 18: Determination of glucose concentration following
degradation of 50 mM cellobiose using 1% lysate in the reaction mixture, measured with the glucose
assay.
The reaction was incubated at 50 °C for 24 hours, and absorbance was recorded at 560 nm. The green
bar represents 50 mM cellobiose as a control, indicating a high background signal.

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                    https://static.igem.wiki/teams/5117/images-wiki/engineering/fig19.png)

Figure 19: Determination of β-glucosidase activity of
pBS0EX-AtBglA, pBS0EX-BhBglA, pBS0EX-PpBglB, pBS2EX-AtBglA, pBS2EX-BhBglA, pBS2EX-PpBglB by using
pNPG as substrate.
The absorbance was measured at 405 nm, representing pNP formation by degradation of pNPG by
different
β-glucosidases. WB800N was used as negative control. Dark purple bars represent uninduced (control)
samples, light purple bars represent induced (0.5 % xylose) samples.

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![](
                    https://static.igem.wiki/teams/5117/images-wiki/engineering/fig20.png)

Figure 20: Determination of the thermostability of spores
displaying β-glucosidase BhBglA with three different linkers (L1, L2, L3) by using pNPG as a
substrate.
Before the reaction with pNPG, the spores were incubated at different temperatures ranging from 40
°C to 90 °C for 2 hours. Following this pre-incubation, the reaction with pNPG was conducted for 10
minutes at 50 °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 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.

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## {{content.reference\_title}}

* 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.
* Chen X., Zaro J. L., Shen, W. C. (2013): Fusion protein linkers: property, design and functionality.
  Advanced
  drug delivery reviews 65(10), 1357-1369.
* 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. doi: 10.1128/MRA.01380-18.
* 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.
* 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.
* Ouyang B., Wang G., Zhang N., Zuo J., Huang Y., Zhao X. (2023): Recent Advances in β-Glucosidase
  Sequence and Structure Engineering. Molecules 28(13).
* Öztel, E. & Mascher T. (2024): unpublished data.
* 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.
* 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.
* Singh N., Sithole B., Govinden R. (2022): Screening for cellulases and preliminary optimisation of
  glucose tolerant β-glucosidase production and characterization. Mycology: An International Journal on
  Fungal Biology 14 (2).
* 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.
* 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.
* The UniProt Consortium , UniProt: the Universal Protein Knowledgebase in 2023, Nucleic Acids Research,
  Volume
  51, Issue D1, 6 January 2023, Pages D523–D531, https://doi.org/10.1093/nar/gkac1052

**BsEglS:**

* MacKay, R M *et al.* “Structure of a *Bacillus subtilis* endo-beta-1,4-glucanase gene.”
  Nucleic acids research vol. 14,22 (1986): 9159-70. doi:10.1093/nar/14.22.9159
* UniProtKB: P10475
  https://www.uniprot.org/uniprotkb/P10475/entry , Accessed: 24.09.2024, 16:29.
* Lindahl, V *et al.* “Nucleotide sequence of an endo-beta-1,4-glucanase gene from *Bacillus
  subtilis* CK-2.” Antonie van Leeuwenhoek vol. 66,4 (1994): 327-32. doi:10.1007/BF00882768 ,
  https://www.ncbi.nlm.nih.gov/gene/938607
* Aa, Kari, *et al.* "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 (1994):
  319-326. https://doi.org/10.1007/BF00882767

**BpEglA:**

* Lima, André OS, *et al.* "Molecular characterization of a β-1, 4-endoglucanase from an endophytic
  *Bacillus pumilus* strain." Applied microbiology and biotechnology 68 (2005): 57-65.
  https://doi.org/10.1007/s00253-004-1740-1 , https://www.ncbi.nlm.nih.gov/protein/AAQ91573 ,
  https://www.ncbi.nlm.nih.gov/nuccore/AY339624
* UniProtKB: Q5YLG1
  https://www.uniprot.org/uniprotkb/Q5YLG1/entry , Accessed: 24.09.2024, 16:31.

**AtCelA:**

* Béguin, P *et al.* “Sequence of a cellulase gene of the thermophilic bacterium *Clostridium
  thermocellum*.”
  Journal of bacteriology vol. 162,1 (1985): 102-5. doi:10.1128/jb.162.1.102-105.1985.
  https://www.ncbi.nlm.nih.gov/protein/AAA83521 , https://www.ncbi.nlm.nih.gov/nuccore/K03088
* UniProtKB: A3DC29
  https://www.uniprot.org/uniprotkb/A3DC29/entry , Accessed: 24.09.2024, 16:30.
* Weng, Zi-Han, *et al.* "Immobilization of recombinant endoglucanase (CelA) from *Clostridium
  thermocellum*
  on modified regenerated cellulose membrane." Catalysts 12.11 (2022): 1356.
  https://doi.org/10.3390/catal12111356
* van der Veen, Douwe *et al.* “Characterization of *Clostridium thermocellum* strains with disrupted
  fermentation end-product pathways.” Journal of industrial microbiology & biotechnology vol. 40,7 (2013):
  725-34.
  doi:10.1007/s10295-013-1275-5
* Schmidt, Andrea *et al.*, Endoglucanase A from *Clostridium thermocellum* at atomic resolution (2001)
  https://doi.org/10.2210/pdb1IS9/pdb
* Schmidt, Andrea *et al.* “Advantages of high-resolution phasing: MAD to atomic resolution.” Acta
  crystallographica. Section D, Biological crystallography vol. 58,Pt 9 (2002): 1433-41.
  doi:10.1107/S0907444902011368
* Kitago, Yu *et al.*, Crystal structure of Cel44A, GH family 44 endoglucanase from *Clostridium
  thermocellum* (2006) https://doi.org/10.2210/pdb2E4T/pdb
* Kitago, Yu *et al.*, Crystal structure of Cel44A, GH family 44 endoglucanase from *Clostridium
  thermocellum* (2007) https://doi.org/10.2210/pdb2EEX/pdb
* Kitago, Yu *et al.*, The crystal structure of Cel44A (2011) https://doi.org/10.2210/pdb2E0P/pdb
* Kitago, Yu *et al.* “Crystal structure of Cel44A, a glycoside hydrolase family 44 endoglucanase
  from
  *Clostridium thermocellum*.” The Journal of biological chemistry vol. 282,49 (2007): 35703-11.
  doi:10.1074/jbc.M706835200,

**AtCelG:**

* UniProtKB: Q05332
  https://www.uniprot.org/uniprotkb/Q05332/entry , Accessed: 24.09.2024, 16:34.
* Lemaire, M and Béguin, P. "Nucleotide sequence of the celG gene of *Clostridium thermocellum* and
  characterization of its product, endoglucanase CelG." Journal of bacteriology 175.11 (1993): 3353-3360.
  https://doi.org/10.1128/jb.175.11.3353-3360.1993 , https://www.ncbi.nlm.nih.gov/protein/CAA49187 ,
  https://www.ncbi.nlm.nih.gov/nuccore/X69390

**AtCelO:**

* Zverlov, Vladimir V *et al.* “A newly described cellulosomal cellobiohydrolase, CelO, from
  *Clostridium
  thermocellum*: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose.”
  Microbiology (Reading, England) vol. 148,Pt 1 (2002): 247-255. doi:10.1099/00221287-148-1-247 ,
  https://www.ncbi.nlm.nih.gov/protein/CAB76938 , https://www.ncbi.nlm.nih.gov/nuccore/AJ275975
* Ahmad, Sajjad *et al.* “Engineering processive cellulase of *Clostridium thermocellum* to divulge the
  role
  of the carbohydrate-binding module.” Biotechnology and applied biochemistry vol. 70,1 (2023): 290-305.
  doi:10.1002/bab.2352
* UniProtKB: Q9L3J2
  https://www.uniprot.org/uniprotkb/Q9L3J2/entry , Accessed: 24.09.2024, 16:35.

**AtCelS:**

* Guimarães, Beatriz G *et al.*, The Crystal Structure and Catalytic Mechanism of Cellobiohydrolase
  CelS,
  the Major Enzymatic Component of the *Clostridium thermocellum* cellulosome (2002)
  https://doi.org/10.2210/pdb1l1y/pdb
* Guimarães, Beatriz G *et al.* “The crystal structure and catalytic mechanism of cellobiohydrolase
  CelS,
  the major enzymatic component of the *Clostridium thermocellum* Cellulosome.” Journal of molecular biology
  vol.
  320,3 (2002): 587-96. doi:10.1016/s0022-2836(02)00497-7
* Kruus, K *et al.* “Exoglucanase activities of the recombinant *Clostridium thermocellum* CelS, a
  major
  cellulosome component.” Journal of bacteriology vol. 177,6 (1995): 1641-4.
  doi:10.1128/jb.177.6.1641-1644.1995
* UniProtKB: A3DH67
  https://www.uniprot.org/uniprotkb/A3DH67/entry , Accessed: 24.09.2024, 16:36.
* Wang, W K *et al.* “Cloning and DNA sequence of the gene coding for *Clostridium thermocellum*
  cellulase
  Ss
  (CelS), a major cellulosome component.” Journal of bacteriology vol. 175,5 (1993): 1293-302.
  doi:10.1128/jb.175.5.1293-1302.1993
* Wilson, Charlotte M., *et al.* "Global transcriptome analysis of *Clostridium thermocellum* ATCC
  27405
  during growth on dilute acid pretreated Populus and switchgrass." Biotechnology for biofuels 6 (2013):
  1-18.
  doi:10.1186/1754-6834-6-179 , https://www.ncbi.nlm.nih.gov/protein/ABN53296
* Wang, W K, and J H Wu. “Structural features of the *Clostridium thermocellum* cellulase SS gene.” Applied
  biochemistry and biotechnology vol. 39-40 (1993): 149-58. doi:10.1007/BF02918985
  https://www.ncbi.nlm.nih.gov/nuccore/L06942

**BhBglA:**

* Naz, S *et al.* “Enhanced production and characterization of a beta-glucosidase from *Bacillus
  halodurans*
  expressed in Escherichia coli.” Biochemistry. Biokhimiia vol. 75,4 (2010): 513-25.
  doi:10.1134/s0006297910040164
* Takami, H *et al.* “Complete genome sequence of the alkaliphilic bacterium *Bacillus
  halodurans* and
  genomic sequence comparison with Bacillus subtilis.” Nucleic acids research vol. 28,21 (2000):
  4317-31.
  doi:10.1093/nar/28.21.4317 , https://www.ncbi.nlm.nih.gov/protein/BAB05642 ,
  https://www.ncbi.nlm.nih.gov/nuccore/BA000004
* UniProtKB: Q9KBK3
  https://www.uniprot.org/uniprotkb/Q9KBK3/entry , Accessed: 24.09.2024, 16:37.

**PpBglB:**

* Tsabitah, Khansa, *et al.* "One-step purification and characterization of β-glucosidase enzyme from
  *Paenibacillus polymyxa*." AIP Conference Proceedings. Vol. 2973. No. 1. AIP Publishing, 2024.
  https://doi.org/10.1063/5.0184761
* Isorna, Pablo *et al.*, beta-glucosidase B from *Paenibacillus polymyxa* (2006)
  https://doi.org/10.2210/pdb2O9P/pdb
* Isorna, Pablo *et al.* “Crystal structures of *Paenibacillus polymyxa* beta-glucosidase B
  complexes
  reveal
  the molecular basis of substrate specificity and give new insights into the catalytic machinery of family
  I
  glycosidases.” Journal of molecular biology vol. 371,5 (2007): 1204-18. doi:10.1016/j.jmb.2007.05.082
* González-Candelas, L *et al.* “Sequences and homology analysis of two genes encoding
  beta-glucosidases
  from *Bacillus polymyxa*.” Gene vol. 95,1 (1990): 31-8. doi:10.1016/0378-1119(90)90410-s ,
  https://www.ncbi.nlm.nih.gov/protein/AAA22264 , https://www.ncbi.nlm.nih.gov/nuccore/M60211
* UniProtKB: P22505
  https://www.uniprot.org/uniprotkb/P22505/entry , Accessed: 24.09.2024, 16:39.

**AtBglA:**

* Gräbnitz, F *et al.* “Structure of the beta-glucosidase gene bglA of *Clostridium
  thermocellum*. Sequence
  analysis reveals a superfamily of cellulases and beta-glycosidases including human lactase/phlorizin
  hydrolase.”
  European journal of biochemistry vol. 200,2 (1991): 301-9. doi:10.1111/j.1432-1033.1991.tb16186.x ,
  https://www.ncbi.nlm.nih.gov/protein/CAA42814 ,
  https://www.ncbi.nlm.nih.gov/nuccore/X60268
* Ahmed, Syed Shoaib *et al.* “Soluble Production, Characterization, and Structural Aesthetics of an
  Industrially Important Thermostable β-Glucosidase from *Clostridium thermocellum* in *Escherichia
  coli*.” BioMed
  research international vol. 2019 9308593. 7 Nov. 2019, doi:10.1155/2019/9308593
* UniProtKB: P26208
  https://www.uniprot.org/uniprotkb/P26208/entry , Accessed: 24.09.2024, 16:42.

**BhrPET:**

* Xi, Xingxiang *et al.* “Secretory expression in *Bacillus subtilis* and biochemical
  characterization of a
  highly thermostable polyethylene terephthalate hydrolase from *bacterium HR29*.” Enzyme and microbial
  technology
  vol. 143 (2021): 109715. doi:10.1016/j.enzmictec.2020.109715
* UniProtKB: A0A2H5Z9R5
  https://www.uniprot.org/uniprotkb/A0A2H5Z9R5/entry , Accessed: 24.09.2024, 16:41.
* Wang, J., Wang, Y.H., HR-PETase from *Bacterium HR29* (2021) https://doi.org/10.2210/pdb7EOA/pdb
* Wang, Ningru, *et al.* "BhrPETase catalyzed polyethylene terephthalate depolymerization: A quantum
  mechanics/molecular mechanics approach." Journal of Hazardous Materials 477 (2024): 135414.
  https://doi.org/10.1016/j.jhazmat.2024.135414
* Kato, Shingo *et al.* “Long-Term Cultivation and Metagenomics Reveal Ecophysiology of Previously
  Uncultivated Thermophiles Involved in Biogeochemical Nitrogen Cycle.” Microbes and environments vol. 33,1
  (2018): 107-110. doi:10.1264/jsme2.ME17165 , https://www.ncbi.nlm.nih.gov/protein/GBD22443

{% endautoescape %}
{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}
{% autoescape false %}

* .
* OUTLINE
* [{{content.experiemnts\_titleA}}
  ![](https://static.igem.wiki/teams/5117/images-wiki/plus-circle.svg)](#submenu1)
  + [Assembly
    ![](https://static.igem.wiki/teams/5117/images-wiki/plus-circle.svg)](#submenu3)
    - [{{content.experiemnts\_title1}}](#Q5)
    - [{{content.experiemnts\_title2}}](#experiments2)
    - [{{content.experiemnts\_title3}}](#experiments3)
    - [{{content.experiemnts\_title4}}](#experiments4)
    - [{{content.experiemnts\_title5}}](#experiments5)
    - [{{content.experiemnts\_title6}}](#experiments6)
    - [{{content.experiemnts\_title7}} *E. coli*
      DH10β](#experiments7)
    - [{{content.experiemnts\_title8}} *E. coli*
      DH10β](#experiments8)
    - [{{content.experiemnts\_title9}} *B.
      subtilis*](#experiments9)
    - [{{content.experiemnts\_title10}}](#experiments10)
    - [{{content.experiemnts\_title11}}](#experiments11)- - [{{content.experiemnts\_title20}}](#experiments20)- [{{content.experiemnts\_title12}}](#experiments12)
          - [{{content.experiemnts\_title13}}](#experiments13)
  + [Qualitative assays for estimation of cellulase activities
    ![](https://static.igem.wiki/teams/5117/images-wiki/plus-circle.svg)](#submenu4)
    - [1. Establishing Qualitative Tests for Cellulase Function](#href_2_1)
    - [2. Background activity screening](#href_2_2)
    - [3. Functioning of the produced recombinant cellulases](#href_2_3)
    - [4. Functioning of the cellulases immobilized on spore surface](#href_2_4)
  + [Quantitative assays for estimation of cellulase activities
    ![](https://static.igem.wiki/teams/5117/images-wiki/plus-circle.svg)](#submenu5)
    - [1. Dinitrosalicylic acid (DNS) assay](#href_3_1)
    - [2. 4-Nitrophenyl acetate (pNPAc) activity assay](#href_3_2)
    - [3. Glucose assay](#href_3_3)
    - [4. 4-Nitrophenyl glucopyranoside activity assay](#href_3_4)
[{{content.experiemnts\_title14}}
![](https://static.igem.wiki/teams/5117/images-wiki/plus-circle.svg)](#submenu2)

+ [{{content.experiemnts\_title15}}](#experiments15)
+ [{{content.experiemnts\_title17}}](#experiments17)
+ [{{content.experiemnts\_title18}}](#experiments18)
+ [{{content.experiemnts\_title19}}](#experiments19)

* [References](#href_references)

---

# **Experiments**

---

## Methods

---

### Assembly

---

#### 1. Amplification of parts by Q5 PCR

Each gene fragment of interest was amplified using a PCR standard protocol.
The components were added according to the following table ([Tab. 1](#tab1)) as suggested from
New England
Biolabs (NEB, United States)
user manual for Q5® High-Fidelity DNA Polymerase.

Table 1: PCR preparation using Q5® High-Fidelity DNA Polymerase
from NEB.

| Component | Stock concentration | 50 µL reaction mix | Final concentration |
| --- | --- | --- | --- |
| 5x Q5® Buffer (New England Biolabs) | 5x | 10 µL | 1x |
| mqH2O (MilliQ water) | - | 32.5 µL | - |
| Forward primer | 10 µM   (10 pmol/µL) | 2.5 µL | 0.5 µM |
| Reverse primer | 10 µM   (10 pmol/µL) | 2.5 µL | 0.5 µM |
| dNTPs (New England BioLabs) | 10 µM (each) | 1 µL | 200 µM |
| Template DNA | - | 1 µL | - |
| Q5® High-Fidelity DNA Polymerase (New England BioLabs) | 2 U/µL | 0.5 µL | 0.02 U/µL |

The general thermocycling conditions for the PCR are shown in the table ([Tab. 2](#tab2))
below.

Table 2: Standard PCR cycling conditions for Q5 PCR (35 cycles from step 2-4).

| Step | Temperature | Duration |
| --- | --- | --- |
| Initial denaturation | 98 °C | 30 s |
| Denaturation | 98 °C | 10 s |
| Annealing | adjusted | 20 s |
| Elongation | 72 °C | 30 s/kb |
| Final extension | 72 °C | 2 min |
| Hold | 15 °C | ∞ |

The used oligonucleotides as well as the construct specific PCR parameters are listed in the
following table ([Tab. 3](#tab3)).
To determine the annealing temperatures, the NEB Tm Calculator was employed.

Table 3: Primer pairs with annealing temperatures and expected
fragment sizes.

| DNA fragment | Template | Primer pair | Primer 5’ - 3’ | Anneal | Size [bp] |
| --- | --- | --- | --- | --- | --- |
| Cellulases and PETase | pSB1C3-gene | oJS\_BBprefix\_fwd | GATCGAATTCGCGGCCGCTTCTAGAG | 72 °C | 1410 (BhBglA), 1413 (PpBglB, AtBglA) |
| oJS\_BBsuffix\_rev | GATCCTGCAGCGGCCGCTACTAGTA | 1566 (BsEglS), 1767 (AtCelG), 2046 (BpEglA), 1500 (AtCelA) |
| 2284 (AtCelS), 2041 (AtCelO) |
| 925 (BhrPET), |
| B0014 - Terminator | pBS1C-P*cotYZ*-sfGFP-cotY | oJS\_BBpre-B0014\_fwd | gatcgaattcgcggccgcttctagagTCACACTGGCTCACCTTC | 65 °C | 146 |
| oJS\_BBsuf-B0014\_rev | gatcctgcagcggccgctactagtaAAATAATAAAAAAGCCGGATTAATAATCTGGC |
| *spec* cassette | pDG1726 | spec-fwd | CAGCGAACCATTTGAGGTGATAGGGACTGGCTCGCTAATAACGTAACGTGACTGGCAAGAG | 72 °C | 1152 |
| spec-rev | CGATACAAATTCCTCGTAGGCGCTCGGCGTAGCGAGGGCAAGGGTTTATTGTTTTCTAAAATCTG |
| *tet* casette | pDG1513 | Tc tet fwd1 | CAGCGAACCATTTGAGGTGATAGGTCTTGCAATGGTGCAGGTTGTTCTC | 68 °C | 2044 |
| Tc rev | CGATACAAATTCCTCGTAGGCGCTCGGGAACTCTCTCCCAAAGTTGATCCC |
| *eglS* up | genomic DNA *B. subtilis* W168 | oJS\_eglS\_up-fwd | GTTATCGCTACAATTGTTAAAAATGC | 60 °C | 1078 |
| oJS\_eglS\_up-rev | CCTATCACCTCAAATGGTTCGCTGGTCAATAACGTAATCAATAAACACG |
| *eglS* down | oJS\_eglS\_down-fwd | CGAGCGCCTACGAGGAATTTGTATCGGGAAAACTGATTTGGGGAACAG | 62 °C | 1084 |
| oJS\_eglS\_down-rev | GGATGTTTCACAGCATATTCAC |
| *bglH* up | oJS\_bglH\_up-fwd | CTTGTGGCAATGATTTTGCTG | 1073 |
| oJS\_bglH\_up-rev | CCTATCACCTCAAATGGTTCGCTGCCCATAAAAATCCTTCTGGAAATCG | 64 °C |
| *bglH* down | oJS\_bglH\_down-fwd | CGAGCGCCTACGAGGAATTTGTATCGCAACTGGTATCAGCAGGTTATC | 1077 |
| oJS\_bglH\_down-rev | GTAACGGACGTTTCTTCAAAAGAC |
| P*cotYZ* | genomic DNA *B. subtilis* W168 | oJS\_(BBpre)-PcotYZ\_fwd | CTAGAAGCTTATCGAATTCGCGGCCGCTTCTAGAGACAGCAACAAATACACTCGTAGC | 61 °C | 238 |
| oJS\_PcotYZ-(RBS-Spac)\_rev | CATTTTTTTTCCTCCTTATTATAGGGTATTTGACTTTAGTCCTTA |
| L1/L3-*cotY* | oEO-CC-cotY-N-fwd | ATGAGCTGCGGAAAAAC | 514 |
| oJS\_cotY-(Spac-Term)\_rev | GGTGAGCCAGTGTGACAGCTTATGATTATCCATTGTGATGATGCTTTTTATC |
| L2-*cotY* | oJS\_(GGGGS4)-cotY\_fwd | ggaggcggtggaagtggtggcggaggtagcATGAGCTGCGGAAAAACC | 62 °C | 544 |
| oJS\_cotY-(Spac-Term)\_rev | GGTGAGCCAGTGTGACAGCTTATGATTATCCATTGTGATGATGCTTTTTATC |
| B0014 - Terminator | pBS1C-P*cotYZ*-sfGFP-cotY | oJS\_(Spac)-Term\_fwd | TCATAAGCTGTCACACTGGCTCACCTTCG | 63 °C | 140 |
| oJS\_Term-(BBsuf)\_rev | CCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTAAAATAATAAAAAAGCCGGATTAATAATCTGG |
| BhBglA-GA | pSB1C3-BhBglA | oJS\_(PcotYZ-RBS-Spac)-BhBglA\_fwd | CCTATAATAAGGAGGAAAAAAAaTGTCGATCA | 1394 |
| oJS\_BhBglA-GA\_rev | GGCCATGGGTTTTTCCGCAGCTCATtgcgccAAGTTCAAAGAACTGATTGGCAATC |
| BhBglA-GGGGS4 | oJS\_(PcotYZ-RBS-Spac)-BhBglA\_fwd | CCTATAATAAGGAGGAAAAAAAaTGTCGATCA | 1418 |
| oJS\_BhBglA-GGGGS4\_rev | ctccgccaccacttccaccgcctccagaacctcctccaccagaacctcctccaccAAGTTCAAAGAACTGATTGGCAATC |
| BhBglA-EAAAK | oJS\_(PcotYZ-RBS-Spac)-BhBglA\_fwd | CCTATAATAAGGAGGAAAAAAAaTGTCGATCA | 1421 |
| oJS\_BhBglA-EAAAK\_rev | GGCCATGGGTTTTTCCGCAGCTCATtcctcctcctttcgccgccgcttctcctcctccAAGTTCAAAGAACTGATTGGCAATC |
| PpBglB-GA | pSB1C3-PpBglB | oJS\_(PcotYZ-RBS-Spac)-PpBglB\_fwd | CCTATAATAAGGAGGAAAAAAAATGAGCGAG | 64 °C | 1397 |
| oJS\_PpBglB-GA\_rev | GGCCATGGGTTTTTCCGCAGCTCATtgcgccAAACCCGTTCTTCGCCATCA |
| PpBglB-GGGGS4 | oJS\_(PcotYZ-RBS-Spac)-PpBglB\_fwd | CCTATAATAAGGAGGAAAAAAAATGAGCGAG | 1421 |
| oJS\_PpBglB-GGGGS4\_rev | ctccgccaccacttccaccgcctccagaacctcctccaccagaacctcctccaccAAACCCGTTCTTCGCCATCA |
| PpBglB-EAAAK | oJS\_(PcotYZ-RBS-Spac)-PpBglB\_fwd | CCTATAATAAGGAGGAAAAAAAATGAGCGAG | 1424 |
| oJS\_PpBglB-EAAAK\_rev | GGCCATGGGTTTTTCCGCAGCTCATtcctcctcctttcgccgccgcttctcctcctccAAACCCGTTCTTCGCCATCA |
| BhrPET-GA | pSB1C3-BhrPETase | oJS\_(PcotYZ-RBS-Spac)-BhrPETase\_fwd | CCTATAATAAGGAGGAAAAAAAATGCAATCAAATCCGTATCAAAGAGGAC | 833 |
| oJS\_BhrPETase-GA\_rev | GGCCATGGGTTTTTCCGCAGCTCATtgcgccTTGGCAATGTCTATTATTTGAGCGA |
| BhrPET-GGGGS4 | oJS\_(PcotYZ-RBS-Spac)-BhrPETase\_fwd | CCTATAATAAGGAGGAAAAAAAATGCAATCAAATCCGTATCAAAGAGGAC | 857 |
| oJS\_BhrPETase-GGGGS4\_rev | ctccgccaccacttccaccgcctccagaacctcctccaccagaacctcctccaccTTGGCAATGTCTATTATTTGAGCGA |
| BhrPET-EAAAK | oJS\_(PcotYZ-RBS-Spac)-BhrPETase\_fwd | CCTATAATAAGGAGGAAAAAAAATGCAATCAAATCCGTATCAAAGAGGAC | 860 |
| oJS\_BhrPETase-EAAAK\_rev | GGCCATGGGTTTTTCCGCAGCTCATtcctcctcctttcgccgccgcttctcctcctccTTGGCAATGTCTATTATTTGAGCGA |
| BsEglS-GA | pSB1C3-BsEglS | oJS\_(PcotYZ-RBS-Spac)-BsEglS\_fwd | CCTATAATAAGGAGGAAAAAAAATGGCAGGGACAAAAACGCCAGTAG | 66 °C | 1466 |
| oJS\_BsEglS-GA\_rev | GGCCATGGGTTTTTCCGCAGCTCATtgcgccATTTGGTTCTGTTCCCCAAATCAG |
| BsEglS-GGGGS4 | oJS\_(PcotYZ-RBS-Spac)-BsEglS\_fwd | CCTATAATAAGGAGGAAAAAAAATGGCAGGGACAAAAACGCCAGTAG | 1490 |
| oJS\_BsEglS-GGGGS4\_rev | ctccgccaccacttccaccgcctccagaacctcctccaccagaacctcctccaccATTTGGTTCTGTTCCCCAAATCAG |
| BsEglS-EAAAK | oJS\_(PcotYZ-RBS-Spac)-BsEglS\_fwd | CCTATAATAAGGAGGAAAAAAAATGGCAGGGACAAAAACGCCAGTAG | 1493 |
| oJS\_BsEglS-EAAAK\_rev | GGCCATGGGTTTTTCCGCAGCTCATtcctcctcctttcgccgccgcttctcctcctccATTTGGTTCTGTTCCCCAAATCAG |
| AtCelO-GA | pSB1C3-AtCelO | oJS\_(PcotYZ-RBS-Spac)-AtCelO\_fwd | CCTATAATAAGGAGGAAAAAAAATGGATGACACTTCTGAAGAACC | 61 °C | 1940 |
| oJS\_AtCelO-GA\_rev | GGCCATGGGTTTTTCCGCAGCTCATtgcgccTTCATTTTGGTTTTCTTCCACC |
| AtCelO-GGGGS4 | oJS\_(PcotYZ-RBS-Spac)-AtCelO\_fwd | CCTATAATAAGGAGGAAAAAAAATGGATGACACTTCTGAAGAACC | 1964 |
| oJS\_AtCelO-GGGGS4\_rev | ctccgccaccacttccaccgcctccagaacctcctccaccagaacctcctccaccTTCATTTTGGTTTTCTTCCACC |
| AtCelO-EAAAK | oJS\_(PcotYZ-RBS-Spac)-AtCelO\_fwd | CCTATAATAAGGAGGAAAAAAAATGGATGACACTTCTGAAGAACC | 1967 |
| oJS\_AtCelO-EAAAK\_rev | GGCCATGGGTTTTTCCGCAGCTCATtcctcctcctttcgccgccgcttctcctcctccTTCATTTTGGTTTTCTTCCACC |
| AtCelS-GA | pSB1C3-AtCelS | oJS\_(PcotYZ-RBS-Spac)-AtCelS\_fwd | CCTATAATAAGGAGGAAAAAAAATGGGTCCTACAAAGGCACCTAC | 62 °C | 2198 |
| oJS\_AtCelS-GA\_rev | GGCCATGGGTTTTTCCGCAGCTCATtgcgccGTTCTTGTACGGCAATGTATC |
| AtCelS-GGGGS4 | oJS\_(PcotYZ-RBS-Spac)-AtCelS\_fwd | CCTATAATAAGGAGGAAAAAAAATGGGTCCTACAAAGGCACCTAC | 2222 |
| oJS\_AtCelS-GGGGS4\_rev | ctccgccaccacttccaccgcctccagaacctcctccaccagaacctcctccaccGTTCTTGTACGGCAATGTATC |
| AtCelS-EAAAK | oJS\_(PcotYZ-RBS-Spac)-AtCelS\_fwd | CCTATAATAAGGAGGAAAAAAAATGGGTCCTACAAAGGCACCTAC | 2225 |
| oJS\_AtCelS-EAAAK\_rev | GGCCATGGGTTTTTCCGCAGCTCATtcctcctcctttcgccgccgcttctcctcctccGTTCTTGTACGGCAATGTATC |

After completion of the PCR reaction, either 5 µl of PCR product were analyzed in a 1 % agarose
gel
and the rest (45 µl)
was purified using the *HiYield® PCR Clean-up/Gel Extraction Kit* (SLG, Germany) or the whole
PCR
product was given
onto the gel and extracted afterwards according to the manufacturer's protocol. The DNA concentration
was measured using the *NanoDrop® ND-1000*
spectrophotometer (Thermo Fisher Scientific Inc., United States) at a wavelength of 260 nm.

---

#### 2. Backbone Phusion PCR

To amplify a target backbone, PCR was performed using Phusion DNA Polymerase (NEB, United States).
The required reaction mixture was prepared and each 50 μl were added to a single PCR tube.
The components were added according to the following table ([Tab.
4](#tab4)) as suggested from
NEBs user
manual
for
Phusion® High-Fidelity DNA Polymerase.

Table 4: PCR preparation using Phusion DNA Polymerase from NEB.

| Components | Stock concentration | 50 µl reaction | Final concentration |
| --- | --- | --- | --- |
| 5X Phusion GC Buffer (NEB) | 5x | 10 µl | 1x |
| mqH2O | - | 31 µl | - |
| Forward Primer | 10 µM (10 pmol/µl) | 2.5 µl | 0.5 µM |
| Reverse Primer | 10 µM (10 pmol/µl) | 2.5 µl | 0.5 µM |
| dNTPs (NEB) | 10 mM of each | 1 µl | 200 µM each |
| DMSO | - | 1.5 µl | 3 % |
| Template DNA | 100 ng | 1 µl | 100 ng |
| Phusion DNA Polymerase (NEB) | 2 U/µl | 0.5 µl | 0.02 U/µl |

The PCR reactions were carried out in the thermocycler according to the program shown
in Table 5:

Table 5: Standard PCR cycling conditions for
Phusion PCR (35 cycles from step 2-4).

| Step | Temperature | Duration |
| --- | --- | --- |
| Initial denaturation | 95 °C | 3 min |
| Denaturation | 95 °C | 20 s |
| Annealing | adjusted | 30 s |
| Elongation | 72 °C | 30 s/kb |
| Finale extension | 72 °C | 10 min |
| Hold | 10 °C | ∞ |

The used oligonucleotides as well as the construct specific PCR parameters are listed in the
following
table ([Tab. 6](#tab6)).
To determine the annealing temperatures, the NEB Tm Calculator was employed.

Table 6: Primer pairs with annealing temperatures and expected fragment sizes.

| Plasmid | Primer pair | Primer 5’ - 3’ | Anneal | Size [bp] |
| --- | --- | --- | --- | --- |
| pSB1C3, pSB1C30 | SB-prep-3P-1 | GCCGCTGCAGTCCGGCAAAAAA | 62 °C | 2043, 2043 |
| SB-prep-2Ea | ATGAATTCCAGAAATCATCCTTAGCG |
| pBS0E-*xylR*-P*xylA*, pBS2E-*xylR*-P*xylA* | RFP-cassette back fwd | TTCGGGTGGGCCTTTCTG | 61 °C | 8398, 8042 |
| XylRPxyl check rev | CTCCTCTTTCTCTAGTATGTGTG |

After the PCR 5 µL of PCR product were analysed in an 1 % agarose gel. To get rid of the
template
plasmid the remaining 45 µL PCR product
were digested using 1 µl of *Dpn*I enzyme (digests methylated DNA). The digest was incubated for 1 hour at
37 °C and after this heat inactivated at 80 °C for 10 minutes.
The product was again analysed in an 1 % agarose gel and then extracted from the gel using the
*HiYield®
PCR Clean-up/Gel Extraction Kit* (SLG).
The DNA concentration was measured using the *NanoDrop® ND-1000* spectrophotometer
(ThermoFisher,
United States) at a wavelength of
260 nm.

---

#### 3. Overlap (Joining) PCR

(protocol generously provided by the lab of Prof. Thorsten Mascher, Faculty of Biology, General
Microbiology, TU Dresden)

##### For gene knockout using a resistance cassette

![](https://static.igem.wiki/teams/5117/images-wiki/overlap-pcr.jpg)

Figure 1: Scheme for the design of an overlap PCR for gene knockout using a
resistance cassette.

The first step in performing the overlap PCR is the selection of the wanted resistance cassette and
the
design of the required primers ([Fig. 1](#fig1)).
The up-forward primer (20 - 25 nt) was designed by selecting a sequence about 1 kb upstream of the
gene or
region to be knocked out.
The up-reverse primer was designed by selecting a sequence within 10 - 50 bp downstream of the start
of
the gene to be knocked out.
At the 5' end of this oligo the corresponding sequence to one end of the cassette was added ([Tab. 7](#tab7)).
The
5' and 3' overhangs are the same
for each cassette. Hence, more than one cassette can be used with one set of up reverse and down
forward
primers.
The down-forward primer was designed by selecting a sequence within 10 - 50 bp upstream of the end of
the
gene to be knocked out.
The sequence corresponding to the other end of the cassette was added to the 5' end of this oligo.
The down-reverse primer (20 - 25 nt) was designed by selecting a sequence about 1 kb downstream of the
gene to be knocked out.

Table 7: 5’ end of joining primers

| 5’ end of joining primer | |
| --- | --- |
| Up-rev | CCTATCACCTCAAATGGTTCGCTG |
| Do-fwd | CGAGCGCCTACGAGGAATTTGTATCG |

The selected cassette as well as the upstream and downstream fragments of the gene to be knocked out
were performed using the Q5 PCR protocol ([see Experiments Q5
PCR](#Q5)).
The cassette was amplified using the appropriate vector and the upstream and downstream fragments were
amplified using *B. subtilis* W168 genomic DNA as template.
The PCR products were purified using the *HiYield® PCR Clean-Up & Gel Extraction Kit*
(SLG,
Germany) and the DNA concentration was measured
using the *NanoDrop® ND-1000* spectrophotometer (ThermoFisher Scientific Inc., United
States)
at a wavelength of 260 nm.
The last step was the joining of the amplified fragments with the cassette by PCR ([Tab. 8](#tab8)).

Table 8: Primer pairs with annealing temperatures and expected fragment sizes.

| DNA-fragment | Primer pair | Primer sequence 5’ - 3’ | Anneal | Size [bp] |
| --- | --- | --- | --- | --- |
| *eglS* up + cassette + *eglS* down | oJS\_eglS\_up-fwd | GTTATCGCTACAATTGTTAAAAATGC | 62 °C | 3264 (*spec*) |
| oJS\_eglS\_down-rev | GGATGTTTCACAGCATATTCAC | 4156 (*tet*) |
| *bglH* up + cassette + *bglH* down | oJS\_bglH\_up-fwd | CTTGTGGCAATGATTTTGCTG | 64 °C | 3252 (*spec*) |
| oJS\_bglH\_down-rev | GTAACGGACGTTTCTTCAAAAGAC | 4144 (*tet*) |

The PCR components were added according to the following table ([Tab. 9](#tab9), modified NEB
user manual for Q5® High-Fidelity DNA Polymerase).
Different ratios (1:2, 1:3) and concentrations (150 ng:300 ng, 10 ng:30 ng, 100 ng: 300 ng) of up/down
fragment to cassette were tested.

Table 9: PCR preparation using Q5® High-Fidelity DNA Polymerase from NEB.

| Components | Stock concentration | 50 µl reaction | Final concentration |
| --- | --- | --- | --- |
| 5X Q5® Reaction Buffer (NEB) | 5x | 10 µl | 1x |
| mqH2O | - | 30.5 µl | - |
| Forward Primer | 10 µM (10 pmol/µl) | 2.5 µl | 0.5 µM |
| Reverse Primer | 10 µM (10 pmol/µl) | 2.5 µl | 0.5 µM |
| dNTPs (NEB) | 10 mM of each | 1 µl | 200 µM each |
| DNA fragment up | variable | 1 µl | variable |
| DNA fragment down | variable | 1 µl | variable |
| Cassette | variable | 1 µl | variable |
| Q5® High-Fidelity DNA Polymerase   (NEB) | 2 U/µl | 0.5 µl | 0.02 U/µl |

The same thermocycler program was used as for the Q5 PCR protocol ([see Experiments Q5
PCR](#Q5)).

After completion of the PCR reaction, 5 μl of PCR product were analyzed in a 1 % agarose gel. The PCR
product was used unpurified or the rest (45 μl) was
purified using the *HiYield® PCR Clean-up/Gel Extraction Kit* (SLG, Germany) according
to the
manufacturer's protocol.
The DNA concentration was measured using the *NanoDrop® ND-1000* spectrophotometer
(Thermo Fisher Scientific Inc., USA) at a wavelength of 260 nm.

##### b) For spore display

The design of the overlap PCR for spore display follows the same principle as the knockout using a
resistance cassette.
It serves to join the four different parts needed for the spore display using *B. subtilis*. These
are the promoter for *cotYZ*, our target gene, the *cotY* gene,
which encodes a spore coat protein, and the terminator. In this order, the parts were added in a 1:3:3:1
ratio for overlap PCR using Q5 DNA polymerase ([Tab. 11](#tab11)).
The ratio was adjusted when the amplification was not efficient.

The used oligonucleotides as well as the construct specific PCR parameters are listed in the following
table ([Tab. 10](#tab10)).
To determine the annealing temperatures, the [NEB Tm
Calculator](https://tmcalculator.neb.com/#!/main) was used.

Table 10: Primer pairs with annealing temperatures and expected fragment sizes.

| DNA-fragment | Primer pair | Primer sequence 5’ - 3’ | Anneal | Size [bp] |
| --- | --- | --- | --- | --- |
| P*cotYZ*-**gene-L3**-*cotY*-Term | oJS\_(BBpre)-PcotYZ\_fwd | CTAGAAGCTTATCGAATTCGCGGCCGCTTCTAGAGACAGCAACAAATACACTCGTAGC | 63 °C | 2283 (BsEglS), 2757 (AtCelO), 3015 (AtCelS), 2211 (BhBglA), 2214 (PpBglB), 1650 (BhrPET) |
| oJS\_Term-(BBsuf)\_rev | CCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTAAAATAATAAAAAAGCCGGATTAATAATCTGG |
| P*cotYZ*-**gene-L3**-*cotY*-Term | oJS\_(BBpre)-PcotYZ\_fwd | CTAGAAGCTTATCGAATTCGCGGCCGCTTCTAGAGACAGCAACAAATACACTCGTAGC | 2337 (BsEglS), 2811 (AtCelO), 3069 (AtCelS), 2265 (BhBglA), 2268 (PpBglB), 1704 (BhrPET) |
| oJS\_Term-(BBsuf)\_rev | CCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTAAAATAATAAAAAAGCCGGATTAATAATCTGG |
| P*cotYZ*-**gene-L3**-*cotY*-Term | oJS\_(BBpre)-PcotYZ\_fwd | CTAGAAGCTTATCGAATTCGCGGCCGCTTCTAGAGACAGCAACAAATACACTCGTAGC | 2310 (BsEglS), 2784 (AtCelO), 3042 (AtCelS), 2238 (BhBglA), 2241 (PpBglB), 1677 (BhrPET) |
| oJS\_Term-(BBsuf)\_rev | CCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTAAAATAATAAAAAAGCCGGATTAATAATCTGG |

The PCR components were added according to the following table ([Tab. 11](#tab11)), modified
NEB user manual for Q5® High-Fidelity DNA Polymerase.

Table 11: PCR preparation using Q5® High-Fidelity DNA Polymerase from NEB.

| Components | Stock concentration | **50 µl reaction** | Final concentration | 50 µl reaction with GC Enhancer |
| --- | --- | --- | --- | --- |
| 5X Q5® Reaction  Buffer (NEB) | 5x | 10 µl | 1x | 10 µl |
| 5X Q5® GC  Enhancer | - | - | - | 5 µl |
| mqH2O | - | 30.5 µl | - | 24.5 µl |
| Forward Primer | 10 µM (10 pmol/µl) | 2.5 µl | 0.5 µM | 2.5 µl |
| Reverse Primer | 10 µM (10 pmol/µl) | 2.5 µl | 0.5 µM | 2.5 µl |
| dNTPs (NEB) | 10 mM of each | 1 µl | 200 µM each | 1 µl |
| Template P*cotYZ* | 10 ng/µl | 1 µl | 0.2 ng/µl | 1 µl |
| Template target gene | 30 ng/µl | 1 µl | 0.6 ng/µl | 1 µl |
| Template cotY | 30 ng/µl | 1 µl | 0.6 ng/µl | 1 µl |
| Template terminator | 10 ng/µl | 1 µl | 0.2 ng/µl | 1 µl |
| Q5® High-Fidelity   DNA Polymerase  (NEB) | 2 U/µl | 0.5 µl | 0.02 U/µl | 0.5 µl |

The same thermocycler program was used as for the Q5 PCR protocol ([see Experiments Q5
PCR](#Q5)).

After PCR, the product was loaded onto a 1 % agarose gel and then extracted from the gel using the
*HiYield® PCR Clean-up/Gel Extraction Kit* (SLG, Germany).
The DNA concentration was measured using the *NanoDrop® ND-1000* spectrophotometer
(Thermo Fisher Scientific Inc., United States) at a wavelength of 260 nm.

---

#### 4. Agarose gel-electrophoresis

Each sample was loaded on to an 1 % agarose gel, containing 2 drops/50 ml agarose ethidium bromide
solution (0.025 %) from Carl Roth.
1 kb Plus DNA Ladder from NEB was used as a ladder. The electrophoresis ran at 120 V. The gel was then
viewed in the UV transilluminator (Fusion FX7 Edge, Vilber, Germany)
under excitation with a wavelength of 260 nm (absorption maximum ethidium bromide). For gel extraction
the desired fragments were cut out of the gel under UV light.
The purification of PCR products and gel extraction were carried out using the *HiYield® PCR Clean-Up
& Gel Extraction Kit* (SLG, Germany) and were performed according
to the manufacturer's protocol. The DNA concentrations of the samples were measured using the
*NanoDrop® ND-1000* spectrophotometer (Thermo Fisher Scientific Inc., United States) at
a
wavelength of 260 nm.

---

#### 5. Restriction

For the cloning of the insert with the respective vector, a restriction digestion of both the vector and
the insert was carried out.
For this purpose, 1 μg of vector and 0.5-1 μg of insert were digested with the respective restriction
enzymes ([Tab. 13](#tab13)).
For the integration of a plasmid into the genome of *B. subtilis*, 1 - 2 μg plasmid were linearized
by using only one restriction enzyme ([Tab. 12](#tab12) and [13](#tab13)).
The restriction with two enzymes was pipetted together as follows:

Table 12: Standard restriction protocol from NEB.

| Components | **20 µl reaction** |
 **50 µl reaction** | Final concentration |
| --- | --- | --- | --- |
| DNA | variable | variable | 1 µg |
| 10x rCutSmart  buffer (NEB) | 2 µl | 5 µl | 1 x |
| Restriction enzyme I | 1 µl | 1 µl | variable |
| Restriction enzyme II | 1 µl | 1 µl | variable |
| mqH2O | Add to 20 µl | Add to 50 µl | - |

After mixing, the reaction mixtures were incubated at 37 °C for 1 hour. The insert and vector were then
purified using the *HiYield® PCR Clean-up/Gel Extraction Kit* (SLG, Germany).
To purify the vector from its insert, three 50 µl reaction tubes were used for gel extraction.
The DNA concentrations were then measured using the *NanoDrop® ND-1000*
spectrophotometer
(ThermoFisher, USA).

The enzymes used for restriction were all supplied by NEB.

\* For the integration of the plasmid into the genome of *B. subtilis* it was linearized
by
using only one restriction enzyme.

Table 13: Restriction enzymes used in this work.

| Vector/ Insert | Enzyme I | Enzyme II |
| --- | --- | --- |
| pBS1C, pSB1C3, pBS0E-*xylR*-P*xylA*, pBS2E-*xylR*-P*xylA* | *Eco*RI-HF | *Pst*I-HF |
| BhBglA, PpBglB, AtBglA, BsBglS, BpBglA, AtCelA, AtCelG, AtCelO, AtCelS, BhrPET | *Eco*RI-HF | *Pst*I-HF |
| P*cotYZ*-enzyme-linker-*cotY*-Term | *Eco*RI-HF | *Pst*I-HF |
| pBS0E-*xylR*-P*xylA*-enzyme | *Spe*I-HF | *Pst*I-HF |
| B0014 Terminator | *Xba*I | *Pst*I-HF |
| pBS2E-*xylR*-P*xylA*-enzyme\* | *Bsa*I-HF | - |
| pBS2E-*xylR*-P*xylA*-enzyme\* | *Sap*I | - |
| pBS1C-P*cotYZ*-enzyme-linker-*cotY*-Term\* | *Bsa*I-HF | - |

---

#### 6. Ligation

Ligation took place after restriction of the vector and the insert. The components were added according
to the following table ([Tab. 14](#tab14)) as suggested from
NEBs user
manual
for T4 DNA Ligase. For this purpose, 50 ng of vector were used with 3, 5 or 7 times the molar excess of
the insert. The molar ratio was determined using the
[NEBioCalculator®](https://nebiocalculator.neb.com/#!/ligation)
for Ligation (NEB).
The preparations were incubated either 20 minutes at 4 °C followed by 2 hours at 16 °C or for 2 hours at
room
temperature.

Table 14: Standard ligation protocol from NEB.

| Component | 20 µl reaction |
| --- | --- |
| T4 DNA Ligase Buffer (10X) (NEB) | 2 µl |
| Vector DNA | 50 ng (0.020 pmol) |
| Insert DNA | variable |
| mqH2O | add to 20 µl |
| T4 DNA Ligase (NEB) | 1 µl |

---

#### 7. Preparation of chemical competent *E. coli* DH10β

*(Mülhardt, C. (2009). Der Experimentator: Molekularbiologie/ Genomics (6. Aufl). Spektrum
Akademischer Verlag.)*

The preparation of chemical competent *E. coli* DH10β was carried out by the technical assistants.

For the preparation of chemical competent *E. coli* DH10β, 500 µl of an overnight culture in 5 mL
LB broth
(37 °C, 180 rpm) was used to inoculate 50 mL LB medium for a day culture.
The day culture was incubated at 37 °C, 180 rpm to an OD600 of 0.4 - 0.7. All following steps
were performed on ice.
After incubation the cells were put on ice for 10 minutes with careful inversion in between. They were
then
centrifuged at 6000 x g, 4 °C for 5 minutes.
The supernatant was removed, and the cell pellet was resuspended in 20 mL TFB1. The cells were then
incubated for 15 minutes on ice with careful inversion in between and again
centrifuged at 6000 x g, 4 °C for 5 minutes. The supernatant was removed, and the cell pellet was
resuspended in 2 mL TFB2 and incubated on ice for 10 minutes.
Each 50 µl competent cells were aliquoted into 1.5 mL tubes and frozen by using liquid nitrogen.
Aliquots were stored at - 80 °C in the freezer.
The composition of the transformation buffers can be found in [Table 15](#tab15).

Table 15: Composition of TFB1 and TFB2.

| Media | Components | Concentration |
| --- | --- | --- |
| TFB1 | CaCl2 | 10 mM |
| Glycerol | 15 % (v/v) |
| Potassium acetate | 30 mM (pH 5.8) |
| RbCl2 | 100 mM |
| MnCl2 | 50 mM |
| TFB 2 | MOPS | 10 mM (pH 7.0) |
| RbCl2 | 10 mM |
| CaCl2 | 75 mM |
| Glycerol | 15 % |

---

#### 8. Transformation of *E. coli* DH10β

*(Protocol generously provided by the lab of Prof. Thorsten Mascher, Faculty of Biology, General
Microbiology, TU Dresden)*

Initially, the chemical competent cells stored at - 80 °C were thawed on ice for 5 - 10 minutes and 4 -
20
μl of ligation mixture or 1 µg of plasmid were added to
50 μl of competent cells. Cells were further incubated on ice for 20 - 30 minutes, followed by heat
shock
at 42 °C for 90 seconds. Immediately after the heat shock,
the cells were incubated on ice for 2 minutes. For cell recovery, 400 μl of LB medium was added and the
samples were incubated at 37 °C for 1 - 2 hours with agitation (550 - 850 rpm)
in a thermomixer. After incubation, the cells were centrifuged (1 minutes, 16,000 x g), around 200 μl of
the
supernatant was discard and the cells were resuspended in the remaining medium.
100 μl of the cells were plated on LB agar (100 μg/ml ampicillin) and incubated overnight at 37 °C.

---

#### 9. Transformation of *B. subtilis*

*(Protocol generously provided by the lab of Prof. Thorsten Mascher, Faculty of Biology, General
Microbiology, TU Dresden)*

For the transformation, 10.43 ml of MNGE medium in a flask were first inoculated with 300 µl fresh
overnight culture of *B. subtilis*
and then incubated at 37 °C, 180 rpm to an OD600 of 1.1 - 1.3. Then 400 μl of the cell
suspension was mixed in a glass tube with (linearized)
1 - 2 μg plasmid or 45 μl PCR product and incubated for one hour at 37 °C, 180 rpm. 400 μl of the cell
suspension, to which no DNA was added,
served as a negative control. Subsequently, 100 μl of expression mix was added to the cells and
incubated for a further hour at 37 °C, 180 rpm.
After incubation the cells were plated on LB selection plates. 100 μl of the negative control were also
plated onto an LB selection plate.
The plates were incubated overnight at 37 °C or for 2 - 3 days at room temperature.
The strains and the introduced constructs can be found on the results page (see Results).

The composition of the media used for *Bacillus* transformation can be found in [Tab. 16](#tab16).

Table 16: Composition of media for *Bacillus* transformation.

| Medium | Components | Volume | Stock concentration |
| --- | --- | --- | --- |
| 10 X MN-Medium | K2HPO4 (x 3 H2O) | 136 g/L | - |
| K2HPO4 | 60 g/L | - |
| Na-citrat (x 2 H2O) | 10 g/L | - |
| MNGE-Medium | 1 X MN-Medium | 9.2 ml | 10 X |
| Glucose | 1 ml | 20 % |
| K-Glutamate | 50 µl | 40 % |
| Fe[III]- ammonium-citrate | 50 µl | 2.2 mg/mL |
| Tryptophan | 100 µl | 5 mg/mL |
| MgSO4 | 30 µl | 1 M |
| Expression Mix | Yeast extract | 500 µl | 5 % |
| Casamino-acids | 250 µl | 10 % |
| H2O | 250µl | - |
| Tryptophan | 50 µl | 5 mg/mL |

---

#### 10. Colony PCR

Colony PCRs were carried out to test potential transformants of *B. subtilis* or *E. coli* on
selection plates. Amplification was carried out using One*Taq*® polymerase.
The required reaction mixture was prepared and each 12 μl were added to a single PCR tube. A few cells
were used as templates, which were taken directly from a colony using
a sterile toothpick and mixed into the reaction mixture of the respective PCR tube.
The master mix contained according to [Table 17](#tab17):

Table 17: PCR preparation using One*Taq*® DNA Polymerase from NEB.

| Components | Stock concentration | **50 µl reaction** | Final concentration |
| --- | --- | --- | --- |
| 5X One*Taq*®   Standard Reaction  Buffer (NEB) | 5x | 10 µl | 1x |
| mqH2O | - | 36.75 µl | - |
| Forward Primer | 10 µM   (10 pmol/µl) | 1 µl | 0.2 µM |
| Reverse Primer | 10 µM   (10 pmol/µl) | 1 µl | 0.2 µM |
| dNTPs | 10 mM of each | 1 µl | 200 µM each |
| Template DNA | - | variable | - |
| One*Taq*® DNA  Polymerase (NEB) | 5 U/µl | 0.25 µl | 0.025 U/µl |

The PCR reactions were carried out in the thermocycler according to the program shown in [Tab. 18](#tab18):

Table 18: Standard PCR cycling conditions for Colony PCR (30 cycles from step 2-4).

| Step | Temperature | Duration |
| --- | --- | --- |
| Initial denaturation | 94 °C | 3 min |
| Denaturation | 94 °C | 20 s |
| Annealing | adjusted | 30 s |
| Elongation | 68 °C | 1 min/kb |
| Finale extension | 68 °C | 5 min |
| Hold | 15 °C | ∞ |

The used oligonucleotides as well as the construct specific PCR parameters are listed in the following
table ([Tab. 19](#tab19)).
To determine the annealing temperatures, the [NEB Tm
Calculator was employed](https://tmcalculator.neb.com/#!/main).

Table 19: Primer pairs with annealing temperatures and expected fragment sizes.

| Construct | Primer pair | Primer 5’ - 3’ | Anneal | Size [bp] |
| --- | --- | --- | --- | --- |
| *E. coli* DH10β | | | | |
| pSB1C3-**enzyme** | seqcheck fwd | TGCCACCTGACGTCTAAG | 47 °C | 1676 (AtBglA, PpBglB), 1673 (BhBglA) |
| seqcheck rev | ATTACCGCCTTTGAGTGA | 1829 (BsEglS), 2030 (AtCelG), 2309 (BpEglA), 1763 (AtCelA) |
| 2555 (AtCelS), 2312 (AtCelO) |
| 1196 (BhrPET) |
| pBS0E-*xylR*-P*xylA*-**enzyme** | oXL\_54 | TGAAAATACTGACGAGGTT | 44 °C | 1564 (AtBglA, PpBglB), 1561 (BhBglA) |
| 1717 (BsEglS), 1918 (AtCelG), 2197 (BpEglA), 1651 (AtCelA) |
| pProEX-1-check-fwd | GGATAACAATTTCACACAGG | 2443 (AtCelS), 2200 (AtCelO) |
| 1084 (BhrPET) |
| pBS2E-*xylR*-P*xylA*-**enzyme** | oXL\_54 | TGAAAATACTGACGAGGTT | 44 °C | 1765 (AtBglA, PpBglB), 1762 (BhBglA) |
| 1918 (BsEglS), 2119 (AtCelG), 2398 (BpEglA), 1852 (AtCelA) |
| pAX01-sacI.selfligated-fw | ctgaaattgatcctccaaac | 2644 (AtCelS), 2401 (AtCelO) |
| 1285 (BhrPET) |
| pBS0E-*xylR*-P*xylA*-**enzyme**-Term | oXL\_54 | TGAAAATACTGACGAGGTT | 44 °C | 1667 (AtBglA, PpBglB), 1664 (BhBglA) |
| 1820 (BsEglS), 2021 (AtCelG), 2300 (BpEglA), 1754 (AtCelA) |
| pProEX-1-check-fwd | GGATAACAATTTCACACAGG | 2546 (AtCelS), 2303 (AtCelO) |
| 1187 (BhrPET) |
| pBS1C-P*cotYZ*-**enzyme-L1**-*cotY*-Term | oEO-OV(3frag)-fwd | CACAAATTAAAAACTGGTCTGATCG | 48 °C | 2275 (PpBglB), 2272 (BhBglA) |
| 2344 (BsEglS) |
| 1711 (BhrPET) |
| pBS1C-P*cotYZ*-**enzyme-L2**-*cotY*-Term | 2329 (PpBglB), 2326 (BhBglA) |
| 2398 (BsEglS) |
| 1C\_rev\_P | TAAGCTGTCAAACATGAGAATTGAC | 2872 (AtCelO) |
| 1765 (BhrPET) |
| pBS1C-P*cotYZ*-**enzyme-L3**-*cotY*-Term | 2302 (PpBglB), 2299 (BhBglA) |
| 2371 (BsEglS) |
| 1738 (BhrPET) |
| ***B. subtilis* WB800N** | | | | |
| *bglH::spec* | oJS\_bglH\_up-fwd | CTTGTGGCAATGATTTTGCTG | 48 °C | 1341 |
| spec-check rev | CGTATGTATTCAAATATATCCTCCTCAC |
| spec-checkfwd | GTTATCTTGGAGAGAATATTGAATGGAC | 49 °C | 1289 |
| oJS\_bglH\_down-rev | GTAACGGACGTTTCTTCAAAAGAC |
| *eglS::tet* | oJS\_eglS\_up-fwd | GTTATCGCTACAATTGTTAAAAATGC | 47 °C | 1517 |
| (tet) tc-check rev | CATCGGTCATAAAATCCGTAATGC |
| *bglH::tet* | oJS\_bglH\_up-fwd | CTTGTGGCAATGATTTTGCTG | 49 °C | 1519 |
| (tet) tc-check rev | CATCGGTCATAAAATCCGTAATGC |
| pBS0E-*xylR*-P*xylA*-**enzyme** | oJS\_BBprefix\_fwd | GATCGAATTCGCGGCCGCTTCTAGAG | 57 °C | 1413 (AtBglA, PpBglB), 1410(BhBglA) |
| 1566 (BsEglS), 1767 (AtCelG), 2046 (BpEglA), 1500 (AtCelA) |
| oJS\_BBsuffix\_rev | GATCCTGCAGCGGCCGCTACTAGTA | 2292 (AtCelS), 2049 (AtCelO) |
| 933 (BhrPET) |
| pBS0E-*xylR*-P*xylA*-**enzyme** | pSET152 checking Fw | CTGCAAGGCGATTAAGTTGGGTAAC | 51 °C | 3044 (AtBglA, PpBglB), 3041 (BhBglA) |
| 3197 (BsEglS), 3398 (AtCelG), 3677 (BpEglA), 3131 (AtCelA) |
| oXL\_2 | TATGACCATGATTACGCCAAGC | 3923 (AtCelS), 3680 (AtCelO) |
| 2564 (BhrPET) |
| pBS2E-*xylR*-P*xylA*-**enzyme** | oJS\_BBprefix\_fwd | GATCGAATTCGCGGCCGCTTCTAGAG | 51 °C | 1951 (AtBglA, PpBglB), 1948 (BhBglA) |
| 2104 (BsEglS), 2305 (AtCelG), 2584 (BpEglA), 2038 (AtCelA) |
| lacA-back-rev | AAGAATCCGCCCATATCGAG | 2830 (AtCelS), 2587 (AtCelO) |
| 1471 (BhrPET) |
| pBS2E-*xylR*-P*xylA*-**enzyme** | lacA-front-fwd | GAACGAAGGGCTAAGAGAAC | 44 °C | 1370 (all enzymes) |
| oXL\_109 | CTGAATACTCGTGTCACT |
| pBS2E-*xylR*-P*xylA*-**BhrPET** | pBS2E int. up fwd | TGCTGCAAAAGAATTTTGTGTCCG | 49 °C | 5301 |
| pBS2E int. up rev | CTTTGCTTTTCATGATTTCATCCC |

---

#### 11. Plasmid isolation

Overnight-cultures of 5 mL LB medium and respective antibiotics were inoculated each with single *E.
coli* colonies grown on selective plates.
The *HiYield® Plasmid Mini DNA Kit* (SLG, Germany) was used for the isolation of plasmid
DNA.
The procedure was carried out according to the manufacturer's protocol.
DNA was eluted with 50 µl MilliQ water and a second elution step was performed to increase yield.
The DNA concentrations of the samples were measured using the *NanoDrop® ND-1000*
spectrophotometer (Thermo Fisher Scientific Inc., United States) at a wavelength of 260 nm.

After concentration determination, samples were prepared for Sanger sequencing, provided by Microsynth
Seqlab GmbH (Germany).

---

#### 12. Expression of secreted and intracellular proteins

To test the desired enzymes, an overnight culture was prepared by inoculating 5 ml of LB medium with 5
µl of Erythromycin (1 mg/ml) and Lincomycin (25 mg/ml).
The culture was incubated overnight at 37 °C with shaking at 180 rpm. The following day, the optical
density at 600 nm (OD600) was adjusted to 0.1 in 20 ml of fresh LB medium with 20 µl of
Erythromycin (1 mg/ml) and Lincomycin (25 mg/ml). Additionally, for experiments with exoglucanases, the
cultures were incubated at 28 °C or 42 °C with shaking at 180 rpm.
The OD was monitored until it reached 0.5 – 0.6, at which point the cultures
were induced with 0.5 % xylose (Carl Roth GmbH + Co. KG, Germany). After induction, the cultures were
left for 24 hours in the incubator under the defined conditions.

To test secreted enzymes, like endoglucanases, exoglucanases and PETases, the cells were centrifuged at
4 °C and 14,000 rpm for 15 minutes. The supernatant (SN) was transferred into a new reaction tube.
The SN was stored short term at 4 °C until further use. Aliquots of the SN were frozen with liquid
nitrogen and stored overnight at - 80 °C. Samples of SN were taken for the sodium dodecyl
sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) to determine the molecular weight of the desired proteins.
To test intracellular enzymes, like β-glucosidases, the cells were centrifuged at 4 °C and 14,000 rpm
for 15 minutes. The SN was discarded, and the pellet was frozen with liquid nitrogen.
The next day, cell disruption buffer with pH 7.5 (50 mM Tris-HCl (Carl Roth GmbH + Co. KG, Germany), 100
mM NaCl (Carl Roth GmbH + Co. KG, Germany)) was used for the cell lysis.
All subsequent steps were performed on ice. 700 µl of disruption buffer along with 7 µl of Protease
Inhibitor Cocktail (100X) (New England Biolabs GmbH, United States) was added to each.
The cells were disrupted by ultrasonication using the Ultrasonic processor Bandelin Sonopuls UW70/
GM70HD with sonotrode SH70 homogeniser (BANDELIN electronic GmbH & Co. KG, Germany).
In sum, 8 - 10 ultrasonic treatments (100 % amplitude, 0.6 cycle, 10 seconds). Between each ultrasonic
treatment cycle, the samples were placed on ice for 15 – 30 seconds.
The success of the disruption was checked by observing less density and coloration in the samples. The
sonication device was wiped with bacillol (Paul Hartmann AG, Germany) and water before switching between
samples.
The suspension was centrifuged at 4 °C and 14,000 rpm for 15 minutes. The crude extract was transferred
to a new reaction tube. The crude extract was stored at 4 °C for short term use and at
- 80 °C for long term storage. Before centrifugation, SDS-PAGE sample of the cell debris were taken.

---

#### 13. SDS-PAGE

SDS-PAGE was conducted to determine the molecular weight of the proteins. The separation gel percentage
varied based on the protein tested (e.g., 8 % for exoglucanases, 10 % for endoglucanases and
β-glucosidases,
12 % for PETases). The gels were prepared following the SDS-PAGE Gel Preparation Kit manual (Abbexa Ltd,
United Kingdom), with tetraacetylethylenediamine (TEMED) and 10 % ammonium persulfate (APS) added just
before proceeding. This was done under the hood, being cautious as the mixture is toxic before
polymerization. Approximately 3 ml of the gel mixture was poured to fill two-thirds of the gel plate,
and isopropanol
was added on top of the separation gel to ensure a smooth, bump-free surface. The stacking gel was
prepared following the manual of the previous named kit, adding TEMED and 10 % APS only when ready to
proceed.
The gel was poured to fill one-third of the gel plate, and the comb was inserted. The gel was left to
set. The glass plates containing the gel were wrapped with a wet towel, placed in a plastic bag, and
stored
in the fridge until needed. Before use, the gel was removed from storage and allowed to warm up to room
temperature.

For sample preparation, 45 µl of sample was mixed with 15 µl of 4X SDS loading buffer (8 % SDS (w/v)
(AppliChem GmbH, Germany), 355 mM β-Mercaptoethanol (Bio-Rad Laboratories, United States)).
The mixture was heated for 10 minutes at 95 °C to ensure protein denaturation. The SDS-PAGE apparatus
(Bio-Rad Laboratories, United States) was assembled, which could hold up to 4 gels, and its leakiness
was checked.
20 µl of samples were loaded into the wells along with 4 µl of the molecular weight marker PageRuler™
Plus Prestained Protein Ladder, 10 bis 250 kDa (Thermo Fisher Scientific Inc., United States). The 1X
SDS
running buffer (diluted from 10X running buffer: Tris base/Glycine/SDS (Bio-Rad Laboratories, United
States)) was added, and the gel was run at a constant 100 V for approximately 1.5 hours. The gel was
placed
in a plastic box and stained using Coomassie Blue solution (1.25 g Coomassie Blue G-250 (Carl Roth GmbH
+ Co. KG, Germany), 250 ml methanol (Carl Roth GmbH + Co. KG, Germany), 50 ml acetic acid (Carl Roth
GmbH + Co.
KG, Germany), 200 ml dH2O) under the hood. The staining process took only 10 - 15 minutes
with
constant shaking. After staining, the Coomassie Blue solution was returned to the bottle for reuse.

For rapid destaining of the gel, isopropanol (Carl Roth GmbH + Co. KG, Germany) was used with three
destaining cycles of 20 minutes with constant shaking. For destaining of the gel overnight, water was
poured
in the box and kept on constant shaking. Once complete, a picture of the gel was taken.

---

#### 14. Spore preparation

To isolate spores, an overnight culture was prepared by inoculating 5 ml of LB medium with 5 µl of
chloramphenicol (5 mg/ml). The culture was incubated overnight at 37 °C with shaking at 180 rpm.
The following day, the optical density at 600 nm (OD600) was adjusted to 0.05 in 5 ml of
fresh LB medium with 5 µl of chloramphenicol (5 mg/ml). This culture was then incubated at 37 °C with
shaking at 180 rpm until it reached the exponential growth phase, which corresponds to an
OD600 between 0.4 and 0.6.

Once the culture reached the desired OD, it was centrifuged at 13,000 g for 30 seconds. The supernatant
was discarded, and the cell pellet was washed with 1 ml of Difco Sporulation Medium (DSM, recipe see
below).
This washing step was repeated, and the cells were resuspended in 4 ml of DSM medium in a sterile glass
tube. The culture was then incubated at 37 °C with shaking at 180 rpm for 24 hours.
In addition to the test strains, the wildtype W168 strain was included as a negative control.

After the 24-hour incubation, the cells were harvested by centrifugation at 13,000 g for 10 minutes. The
supernatant was discarded, and the cell pellet was resuspended in 1 ml of dH2O. To lyse the
cells,
1.5 µl of lysozyme (75 µg/ml, Carl Roth GmbH + Co. KG, Germany) was added, and the tubes were gently
inverted before being incubated at 37 °C for 1 hour.

Following cell lysis, the culture was centrifuged again using the same settings. After centrifugation, a
series of washing steps were performed. First, the cells were washed with 2 ml of dH2O,
followed by centrifugation. After discarding the supernatant, the cells were resuspended in 2 ml of 0.05
%
SDS to remove organic residues. After another centrifugation step, the cells were washed
three more times with 2 ml of dH2O. Finally, the pellet was resuspended in 1 ml of
dH2O and stored in the dark at room temperature.

The OD of the spore suspension was measured using a 1:10 dilution (900 µl of dH2O and 100 µl
of spore solution). The OD was then adjusted by fixing the OD to 2 for the glucose assay, DNS assay, and
assays that use pNPG or pNPAc as substrate with a final OD concentration of 0.2 in the reaction. For the
qualitative plate assays, the OD was fixed to 0.2 for to test endoglucanases and exoglucanases.
Different OD were used to observe the enzymatic reaction of exoglucanase displaying spores on plates
with PASC-Agar and PASA-Agar.

---

### Qualitative assays for estimation of cellulase activities

---

#### 1. Establishing Qualitative Tests for Cellulase Function

To evaluate the activity of β-glucosidase, LB-Agar plates containing Esculin (Carl Roth GmbH + Co.
KG, Germany, recipe see in [Table 24](#tab25)) were
prepared.
Wells approximately 0.5 cm
in diameter were created in the agar.
Into these wells, 10 μl of Accellerase 1500 enzyme mix (Genencor International, United States) was
added, both in undiluted form and in dilutions of 1:10 and 1:100. The plates were incubated at 50 °C
for 6 hours.

For testing endoglucanase and exoglucanase activities, LB-Agar plates with Carboxymethyl cellulose
(CMC, Carl Roth GmbH + Co. KG, Germany) and Avicel (Carl Roth GmbH + Co. KG, Germany) were used,
respectively.
The media components are described in [Table 24](#tab25).
Like the
β-glucosidase assay,
10 μl of the undiluted Accellerase 1500 enzyme mix and its dilutions (1:10 and 1:100) were pipetted
into the wells.
The plates were incubated at 50 °C for varying time periods, ranging from 0 to 24 hours. After
incubation, the plates were stained with a 0.1 % (w/v) congo red solution (Sigma-Aldrich®,
United
States) at room
temperature for 15 minutes. After discarding the congo red, a 1 M NaCl solution was used for
destaining for 15 minutes, which was also subsequently discarded.

Additionally, for determining exoglucanase activity, LB-Agar-Avicel plates with 0.5 % (w/v) avicel were
prepared. 20 μl of Accellerase 1500 solution was added to the wells in the LB-Agar-Avicel media with
avicel end concentration of 0.5 % or 1 % respectively. Then, the plates were incubated for 48 h at 37 °C
and stained with congo red method as described above.

Furthermore, the exoglucanase activity was examined using cellulose overlay assay. For that 10 μl of
Accellerase 1500 mix was pipetted onto LB agar plates.
After the suspension dried out, cellulose overlay ([Table 24](#tab25)) was poured onto the
medium. The plates were incubated at 37 °C for 24 h.
Then they were stained with congo red method as described above.

Finally, PASC-Agar and PASA-Agar plates ([Table 24](#tab25)) were also used for exoglucanase
activity determination.
In these assays, 20 μl of commercially acquired cellobiohydrolase I from *Hypocrea jecorina*
(Sigma-Aldrich®, United States) was pipetted into the wells.
The plates were then incubated at 50 °C for 24 hours.

The protocol for preparing phosphoric acid swollen cellulose (PASC) and phosphoric acid swollen
Avicel (PASA) is based on the method described by [Zhang *et al.*](https://pubs.acs.org/doi/10.1021/bm050799c?src), (2006).
To begin, 0.2 g of cellulose (microcrystalline, powder, 20 μm, Sigma-Aldrich®, United States)
or
Avicel was weighed into a 50-mL centrifuge tube. Then, 600 μL of distilled water (dH2O)
was added to suspend the
substrate. Following this, 10 mL of ice-cold 85 % phosphoric acid (H3PO4, Carl
Roth GmbH + Co. KG,
Germany) was slowly added to the suspension. The mixture was briefly vortexed before and after the
addition of
the H3PO4 to ensure proper mixing.

After this step, the suspension was incubated on ice for 1 hour with occasional vortexing. Once the
incubation period was complete, 30 mL of ice-cold dH2O was added to the tube, and the
mixture was
vortexed again.
The suspension was then centrifuged at 5000 g and 4°C for 20 minutes, and the supernatant was
carefully removed.

The pellet was washed four times by resuspending 40 mL of dH2O, centrifuging, and
discarding the supernatant after each wash. After the final wash, 500 μL of 2 M
Na2CO3 as added, followed by 2 – 3 additional
washing steps with 50 mM phosphate buffer (pH 7) until the pH of the PASC/PASA reached 7 upon
resuspension in buffer.

Images of all plates were taken with P.CAM360 (software PlayMemories Mobile).

---

#### 2. Background activity screening

For the background activity screening, overnight cultures were prepared using the following
*B. subtilis* strains: BKE18130, BKE03410, BKE40110, BKE39070, BKE38560, BKE39260, W168, and
WB800N, along with
*E. coli* as a negative control. For BKE strains, antibiotics were added (Erythromycin (1
mg/ml) and Lincomycin (25 mg/ml)). Each overnight culture was adjusted to an OD600 of
0.5, and 10 μL of each solution
was pipetted onto the respective LB agar medium containing CMC, Avicel, or esculin as substrates.
These media were prepared as described previously for testing cellulase activities, with or without
the addition
of 0.5 % xylose to induce enzyme production. The plates were incubated at 37 °C for various time
periods, ranging from 0 to 24 hours, to observe potential cellulase activity.

---

#### 3. Functioning of the produced recombinant cellulases (intracellular and secreted)

Initially, to test the functioning of the intracellularly produced β-glucosidases LB-Agar-Esculin
with and without 0.5 % xylose was used. 10 μl of each solution containing overnight cultures with
OD600 of 0.5
were pipetted onto the medium. The plates were incubated 24 hours at 37 °C.

For evaluating the functionality of secreted endoglucanases, CMC-Agar plates were used. In these
tests, 15 μL of supernatant containing the respective endoglucanases was applied as the sample.
The supernatant from *B. subtilis* WB800N was used as a negative control, while a 10 μL aliquot
of Accellerase 1500 enzyme mix diluted 1:100, was used as a positive control. The plates were
incubated at 50 °C
for 24 hours and subsequently stained with congo red solution as previously described.

To test exoglucanase activity, PASC-Agar and PASA-Agar plates were used. For these assays, 70 μL of
supernatants, with and without the respective exoglucanases, were applied to the plates.
The plates were incubated for 24 hours at 50 °C to evaluate the enzyme activity.

---

#### 4. Functioning of the cellulases immobilized on spore surface

The OD of the spore suspension was measured using a 1:10 dilution (900 µl of dH2O and 100
µl of
spore solution). For the qualitative plate assays, the OD was fixed to 0.2 for to test
endoglucanases and exoglucanases.
Different OD were used to observe the enzymatic reaction of exoglucanase displaying spores on plates
with PASC-Agar and PASA-Agar.

---

### Quantitative assays for estimation of cellulase activities

---

#### 1. Dinitrosalicylic acid (DNS) assay

DNS assay protocol was adapted, with modifications, from the Environmental Biotechnology lab of the
International Institute Zittau, provided by Prof. Dr. Christiane Liers. At first, DNS stop solution
was prepared.
1 g of DNS (Sigma-Aldrich®, United States) was dissolved in 50 ml dH2O under
stirring at
50 °C. Then 10 ml of 4 M NaOH/KOH solution were added drop by drop. Finally, 30 g of Rochelle salt
(potassium sodium tartrate
tetrahydrate, Sigma-Aldrich®, United States) were given into the solution under stirring and
the
volume was adjusted with dH2O to 100 ml. The stop solution was stored at room temperature
in the dark.

Calibration standards with concentrations summarized in [Table
20](#tab20) were prepared
for glucose and cellobiose (Carl Roth GmbH + Co. KG, Germany) calibration curves. 700 μl of the
calibration standards were
mixed with 700 μl DNS stop solution, incubated at 99 °C in a heat block for 5 minutes and then
cooled
down on ice. 100 μl of the resulted solution were added to 900 μl of 50 mM phosphate buffer with pH
7 in a
cuvette and the absorption was measured at 540 nm with UV-VIS spectrophotometer UV-1280 (Shimadzu,
Japan).

Table 20: Concentrations of glucose and cellobiose in calibration standards applied for
construction of the calibration curve.

| calibration standard | c(standard), mM | c(glucose), µg/ml | c(cellobiose), µg/ml |
| --- | --- | --- | --- |
| 1 | 11.1 | 2000 | 3800 |
| 2 | 8.88 | 1600 | 3040 |
| 3 | 6.66 | 1200 | 2280 |
| 4 | 4.44 | 800 | 1520 |
| 5 | 2.22 | 400 | 760 |
| 6 | 1.11 | 200 | 380 |

##### a) Tests with supernatants containing endoglucanases (BsEglS, BpEglA, AtCelA, AtCelG)

A reaction mixture consisted of

* 650 µl of 1 % CMC solution prepared in 50 mM phosphate buffer,
* 50 μl of endoglucanase containing supernatants or controls.

The reaction mixtures were incubated at 50 °C for varying times: 1 hour, 5 hours, or 13 hours. As a
negative control, 50 mM phosphate buffer (pH 7) was used.

After the incubation period, 700 µL of DNS stop solution was added to each reaction, followed by
heating the mixture for 5 minutes at 99 °C. The reaction mixtures were then cooled on ice.
From each cooled solution, 100 µL was taken, mixed with 900 µL of phosphate buffer in a cuvette, and
the absorbance at 540 nm (A540) was measured to estimate the enzyme activity.

##### b) Initial activity tests with BsEglS immobilized on spore surface

The OD of the spore suspension was measured using a 1:10 dilution (900 µl of dH2O and 100
µl of spore solution).
The OD was then adjusted by fixing the OD to 2.8 for DNS assay with a final OD concentration of 0.2
in the reaction.

A reaction mixture was prepared with

* 650 µl of 1 % CMC solution prepared in 50 mM phosphate buffer,
* 50 μl of endoglucanase containing supernatants or controls.

The reaction mixture was incubated at 50 °C for either 24 hours or 30 minutes, depending on the
experimental conditions. After the incubation period, 700 µL of DNS solution was added to each
sample,
and the mixture was heated at 99 °C for 5 minutes. Following heating, the mixture was cooled on ice
and then centrifuged at 13,000 g for 1 minute. The solution was diluted 1:10, and absorbance was
measured at 540 nm.

##### c) Optimal temperature of BsEglS-L1,BsEglS-L2, BsEglS-L3

The optimal temperature for BsEglS-L1,BsEglS-L2, BsEglS-L3 was determined by conducting the assay as
previously
described (see point [(b)](#b1)) for 30 minutes, but at
varying
temperatures ranging from
40 °C to 90 °C.
This allowed for the assessment of enzyme activity at different temperatures to determine the
temperature at which the enzyme exhibits maximal activity.

##### d) Thermostability determination of BsEglS-L1,BsEglS-L2, BsEglS-L3

To determine the thermostability of BsEglS-L1,BsEglS-L2, BsEglS-L3, the assay was conducted as described
in point
(b) for 30 minutes. However, before performing the assay, spore solutions were incubated at various
temperatures
ranging from 40 °C to 90 °C for 2 hours. After the heat treatment, the samples were allowed to cool
down to room temperature. Following this pre-incubation, the assay was carried out to assess the
residual enzyme
activity.

---

#### 2. 4-Nitrophenyl acetate (pNPAc) activity assay

The PETase activity assay was adapted from the method described by Xi *et al.* ([Xi
*et al.*, 2020](https://www.sciencedirect.com/science/article/pii/S0141022920302088?via=ihub#sec0095)). A fresh stock solution of 20 mM 4-nitrophenyl acetate (pNPAc) (Thermo Fisher
Scientific, United States) in
dimethyl sulphoxide (DMSO) (Carl Roth GmbH + Co. KG, Germany) was prepared immediately before the
experiments. The stock solution was not stored for more than one day to ensure reagent stability and
assay accuracy.

##### a) Activity tests with supernatants containing BhrPET

A 100 µl reaction mixture was prepared as follows:

* 20 µL of 250 mM sodium phosphate buffer (pH 7.0) to achieve a final concentration of 50 mM
  in the reaction,
* 5 µL of 20 mM 4-nitrophenyl acetate (pNPAc) solution in DMSO, resulting in a final substrate
  concentration of 1 mM and 5 % DMSO in the reaction,
* x µL of supernatant containing BhrPET, and
* x µL of dH2O to bring the final volume to 100 µL.

The reaction was initiated by adding the supernatant and incubated at 40 °C. A continuous absorption
measurement at 405 nm was carried out for 10 minutes using a TECAN Microplate Reader Infinite®
200
PRO.

##### b) Activity tests with BhrPET immobilized on spore surface

A 400 µl reaction mixture was prepared in a 1.5 ml Eppendorf tube consisting of:

* 80 µl of 250 mM phosphate buffer (pH 7),
* 40 µl of spore solution (OD = 2),
* 20 µl of 20 mM pNPAc,
* 260 µl of dH2O.

The mixture was incubated at 40 °C for 10 minutes, followed by centrifugation at 13,000 g for
1 minute.
After centrifugation, 100 µl of the supernatant was transferred into a 96-well plate, and absorbance
was measured at 405 nm.

Additionally, the described assay was carried out with BhBglA immobilized on spore surface to test the
interaction of spores with pNPAc.

##### c) Optimal temperature of BhrPET-L1

The optimal temperature for BhrPET-L1 was determined by conducting the assay as previously described
(see point ([b](#b2))) for 10 minutes, but at varying
temperatures
ranging from 40 °C to
90 °C.
This allowed for the assessment of enzyme activity at different temperatures to determine the
temperature at which the enzyme exhibits maximal activity.

##### d) Thermostability determination of BhrPET-L1

To determine the thermostability of BhrPET-L1 the assay was conducted as described in point ([b](#b2)) for 10 minutes. However, before performing the assay, spore solutions were
incubated at various temperatures
ranging from 40 °C to 90 °C for 2 hours. After the heat treatment, the samples were allowed to cool
down to room temperature. Following this pre-incubation, the assay was carried out to assess the
residual
enzyme activity.

---

#### 3. Glucose assay

To determine the glucose concentration in a reaction, Amplex™ Red Glucose/Glucoseoxidase-Assay-Kit
(Thermo Fisher Scientific Inc., United States) was used.

First, all solutions were prepared according to the kit manual. To prepare the 1X Reaction Buffer
(RB), 4 mL of 5X RB (Component C, white cap) were added to 16 ml of dH2O. A 10 U/ml stock
solution of
horseradish peroxidase (HRP) was prepared by dissolving the contents of the vial of HRP (Component
D, yellow cap) in 1 ml of 1X RB. The solution was divided into single-use aliquots and stored frozen
at –20 °C.
To prepare a 100 U/ml glucose oxidase stock solution, the contents of the glucose oxidase (Component
E, orange cap) were dissolved in 1 mL of 1X RB. This stock was also divided into single-use aliquots
and stored
frozen at –20 °C. For the 10 mM stock solution of Amplex® Red reagent, the vial (Component A,
blue
cap) and DMSO (Component B, green cap) were allowed to warm to room temperature. Just before use,
the contents of
the Amplex® Red reagent vial were dissolved in 60 µL of DMSO.

Next, a working solution containing 100 µM Amplex® Red reagent, 0.2 U/mL HRP, and 2 U/ml
glucose
oxidase was prepared by mixing the following: 50 µl of the 10 mM Amplex® Red reagent stock
solution,
100 µl of the 10 U/ml HRP stock solution, 100 µl of the 100 U/mL glucose oxidase stock solution and
4.75 ml of 1X Reaction Buffer.

To generate a glucose standard curve, an appropriate amount of glucose was dissolved in 1X RB to
produce concentrations of 0 to 150 µM, each in a volume of 50 µL. The final glucose concentrations
in
the reaction were twofold lower (i.e., 0 to 100 µM). 50 µL of each standard curve sample was loaded
into a 96-well plate.

After loading the samples (50 µL) into the 96-well plate, 50 µL of the Amplex® Red
reagent/HRP/glucose oxidase working solution was added to each well.
The reactions were incubated at room temperature for 30 minutes, protected from light.
Afterward, absorbance was measured at 560 nm using a TECAN Microplate Reader Infinite® 200
PRO.

For sample preparation (lysate or spore solution), we followed the respective protocols for induced
expression or spore preparation. To quantify the glucose produced by cellobiose degradation
dissolved in
1X RB, the following reactions were set up prior to the glucose assay and incubated for varying time
periods (up to 24 hours).

##### a) Activity tests with lysates containing β-glucosidases (BhBglA, PpBglB, AtBglA)

A 400 µl reaction mixture was prepared with the following components:

* 160 µl of 125 mM cellobiose, yielding a final concentration of 50 mM in the reaction,
* x µl of lysate containing glucosidases,
* x µl of 1X RB to complete reaction volume.

The mixture was incubated at 50 °C for varying time periods (up to 24 hours). After incubation, the
reaction was terminated by heating the samples at 75 °C for 15 minutes, followed by centrifugation
at 5,000 g
for 1 minute. Subsequently, 50 µl of the working solution from the glucose assay kit was added, and
the mixture was incubated in the dark for 30 minutes. Absorbance was measured at 560 nm.

##### b) Optimization of glucose assay with lysates

To optimize the glucose assay performance with lysates, varying percentages of lysate from WB800N were
tested out.
The samples were mixed with 50 mM cellobiose, 200 µM glucose, and 1X reaction buffer from the
Amplex® Red Glucose/Glucose Oxidase Assay Kit.
Glucose concentration was measured using the kit, and absorbance was recorded at 560 nm.

Additionally, cellobiose purification protocol was tested out ([McCarthy et al, 2004](https://doi.org/10.1074/JBC.M305642200) ).
Cellobiose was treated with glucose oxidase for 3 hours at 37 °C. The reaction was terminated by the
subsequent incubation at 85 °C.
The remaining impurities were removed by filtration performed prior to β-glucosidase reaction.

##### c) Activity tests with β-glucosidases displaying spores (BhBglA, PpBglB)

A 400 µl reaction mixture was prepared as follows:

* 160 µl of 125 mM cellobiose, yielding a final concentration of 50 mM in the reaction,
* 40 µl of spore solution (OD = 2),
* 200 µl of 1X RB.

The mixture was incubated at 50 °C for varying time periods (up to 24 hours). After incubation, the
reaction was centrifuged at 13,000 rpm for 1 minute. As with the lysate samples, 50 µl of the
working
solution from the glucose assay kit was added, and the mixture was incubated in the dark for 30
minutes. Absorbance was measured at 560 nm.

---

#### 4. 4-Nitrophenyl glucopyranoside activity assay

The activity of β-glucosidase was measured by assessing the hydrolysis of
*p*-nitrophenyl-β-D-glucopyranoside (pNPG) (Merck Millipore, United States), using the initial rate
of
colored reaction product accumulation,
as described by [Korotkova *et al.*](https://pubmed.ncbi.nlm.nih.gov/19538132/) (2009).
The enzyme samples (either lysate or spore solution) were mixed with 5 mM pNPG substrate in 50 mM
sodium phosphate buffer (pH 7.0). After incubation at 50 °C for 10 minutes,
the reaction was terminated by adding 0.5 M Na2CO3. (Carl Roth GmbH + Co. KG,
Germany). The release of *p*-nitrophenol (pNP), indicated by a yellow color,
was measured using a TECAN Microplate Reader Infinite® 200 PRO at 405 nm.

##### a) Activity tests with lysates containing β-glucosidases (BhBglA, PpBglB, AtBglA)

A reaction mixture was prepared by mixing 20 µL of β-glucosidase lysate (BhBglA, PpBglB, AtBglA)
with 180 µL of 5 mM pNPG substrate, dissolved in 50 mM sodium phosphate buffer (pH 7.0).
The mixture was incubated at 50 °C for 10 minutes to allow the reaction to occur. To stop the
reaction, 100 µl of ice-cold 0.5 Na2CO3 was added.

##### b) Activity tests with β-glucosidases displaying spores (BhBglA-L1, BhBglA-L2 and BhBglA-L3, PpBglB-L1, PpBglB-L3 )

A reaction mixture was prepared by mixing 40 µL of β-glucosidase-displaying spore solution (BhBglA-L1, BhBglA-L2 and BhBglA-L3, PpBglB-L1, PpBglB-L3) with an OD of 2, with 360 µL of 5 mM pNPG substrate, dissolved in 50 mM sodium citrate
buffer (pH 7.0).
The mixture was incubated at 50 °C for 10 minutes to allow the reaction to proceed. The reaction was
then terminated by adding 200 µl of ice-cold 0.5 M Na2CO3.

##### c) Optimal temperature of BhBglA-L1, BhBglA-L2 and BhBglA-L3

The optimal temperature for BhBglA-L1, BhBglA-L2 and BhBglA-L3 was determined by conducting the assay as previously
described (see point ([b](#b3))) for 10 minutes, but at
varying
temperatures ranging from
40 °C to 90 °C.
This allowed for the assessment of enzyme activity at different temperatures to determine the
temperature at which the enzyme exhibits maximal activity.

##### d) Thermostability determination of BhBglA-L1, BhBglA-L2 and BhBglA-L3

To determine the thermostability of BhBglA-L1, BhBglA-L2 and BhBglA-L3, the assay was conducted as described in point
([b](#b3)) for 10 minutes. However, before performing the
assay,
spore solutions were
incubated at various
temperatures ranging from 40 °C to 90 °C for 2 hours. After the heat treatment, the samples were
allowed to cool down to room temperature. Following this pre-incubation, the assay was carried out
to assess
the residual enzyme activity.

##### e) Reusability of the spore-displayed β-glucosidases

To examine the reusability of the spore-displayed enzymes for BhBglA with three different linkers the
spores were used in five reaction cycles, with a washing step between each cycle.
The number of spores in the first cycle was adjusted to achieve an OD600 of 0.2 in the
reaction mixture. The washing step involved removing the reaction products by ensuring the spores
settled at the bottom of the reaction tube through centrifugation for 5 minutes at 13,000 rpm. The
supernatant was discarded, followed by the addition of 1 ml of dH2O, another
centrifugation step, and subsequent removal of the water. 100 µl of fresh dH2O were added,
and the spores were stored until the next usage (20 minutes later).
The reaction was conducted with pNPG as the substrate for 15 minutes instead of the usual 10 minutes.
After completing the final fifth cycle, the reaction mixture was measured,
then incubated for an additional 1 hour, followed by another measurement.

---

## Materials

---

### Reagents, enzymes and kits

Molecular biological agents and enzymes as well as molecular biological kits used during our project
are listed in [Tab. 21](#tab21), [Tab. 22](#tab22) and [Tab. 23](#tab23).

Table 21: Molecular biological reagents.

| Reagent | Manufacturer |
| --- | --- |
| 1 kb Plus DNA Ladder | New England BioLabs Inc., United States |
| Deoxynucleoside Mix (dNTPs) | New England BioLabs Inc., United States |
| DMSO (100 %) | New England BioLabs Inc., United States |
| Gel Loading Dye Purple (6X) | New England BioLabs Inc., United States |
| One*Taq*® Standard Reaction Buffer (5X) | New England BioLabs Inc., United States |
| Phusion® GC Reaction Buffer (5X) | New England BioLabs Inc., United States |
| Q5® GC Enhancer (5X) | New England BioLabs Inc., United States |
| Q5® Reaction Buffer (5X) | New England BioLabs Inc., United States |
| rCutsmart® Buffer (10X) | New England BioLabs Inc., United States |
| T4 DNA Ligase Buffer (10X) | New England BioLabs Inc., United States |

Table 22: Enzymes.

| Enzyme | Units | Manufacturer |
| --- | --- | --- |
| Lysozyme | 20.000 U/mg | Carl Roth GmbH + Co. KG, Germany |
| One*Taq*® DNA Polymerase | 5.000 U/ml | New England BioLabs Inc., United States |
| Q5® High-Fidelity DNA Polymerase | 2.000 U/ml | New England BioLabs Inc., United States |
| Restriction enzymes | | |
| *Bsa*I-HF® | 20.000 U/ml | New England BioLabs Inc., United States |
| *Eco*RI-HF® | 20.000 U/ml |
| *Sap*I | 10.000 U/ml |
| *Spe*I-HF® | 20.000 U/ml |
| *Pst*I-HF® | 20.000 U/ml |
| *Xba*I | 20.000 U/ml |
| T4 DNA Ligase | 400.000 U/ml | New England BioLabs Inc., United States |
| Endoglucanase from  *Acidothermus cellulolyticus* | 2.0 U/mg | Sigma-Aldrich®, United States |
| Cellobiohydrolase I from *Hypocrea jecorina* | 0.13 U/mg | Sigma-Aldrich®, United States |
| Accellerase 1500 enzyme mix | Endoglucanase activity: 2200 – 2800 CMC U/g | Genencor International, United States |
| β-Glucosidase activity: 525 – 775 pNPG U/g |

Table 23: Molecular biological kits.

| Purpose | Kit | Manufacturer |
| --- | --- | --- |
| DNA purification | HiYield® PCR Clean-up/Gel Extraction Kit | Süd-Laborbedarf GmbH |
| Plasmid isolation | HiYield® Plasmid Mini DNA Kit | Süd-Laborbedarf GmbH |

---

### Media und supplements

Table 24: Media.

| Medium | Composition | Manufacturer |
| --- | --- | --- |
| DSM medium | 8 g Nutrient broth | Carl Roth GmbH + Co. KG, Germany |
| 1 g KCl | Carl Roth GmbH + Co. KG, Germany |
| 1 ml MgSO4 (1 M) | Grüssing GmbH, Germany |
| 1 ml MnCl2 (10 mM) | Sigma-Aldrich® Chemie GmbH, Germany |
| 1000 ml dH2O |  |
| After autoclaving: |  |
| 1 ml FeSO4 (1 mM) | Caesar & Loretz GmbH, Germany |
| 0.5 ml CaCl2 (1 M) | Honeywell Specialty Chemicals Seelze GmbH, Germany |
| Expression mix | 500 µl Yeast extract (5 %) | Carl Roth GmbH + Co. KG, Germany |
| 250 µl Cas amino acids (10 %) | Thermo Fisher Scientific, United States |
| 250 µl dH2O |  |
| 50 µl Tryptophan (5 mg/ml) | Carl Roth GmbH + Co. KG, Germany |
| LB agar | 40 g LB-Agar (Luria/Miller) \*Dehydrated commercial culture media | Carl Roth GmbH + Co. KG, Germany |
| 1000 ml dH2O |  |
| LB medium | 25 g LB-Broth (Luria/Miller) \*Dehydrated commercial culture media | Carl Roth GmbH + Co. KG, Germany |
| MNGE medium | 8.28 ml dH2O |  |
| 920 µl MN medium (10X) |  |
| 1 ml Glucose (20 %) | Carl Roth GmbH + Co. KG, Germany |
| 50 µl Potassium Glutamate (40 %) | Sigma-Aldrich®, United States |
| 50 µl Iron(III) ammonium citrate (2,2 mg/ml) |  |
| 100 µl Tryptophan (5 mg/ml) | Carl Roth GmbH + Co. KG, Germany |
| 30 µl MgSO4 (1 M) | Grüssing GmbH, Germany |
| Starch agar | 7.5 g Nutrient broth | Carl Roth GmbH + Co. KG, Germany |
| 5 g Starch | Thermo Fisher Scientific, United States |
| 15 g Agar | Carl Roth GmbH + Co. KG, Germany |
| 1000 ml dH2O |  |
| LB-Agar-Esculin | 40 g LB-Agar (Luria/Miller) \*Dehydrated commercial culture media | Carl Roth GmbH + Co. KG, Germany |
| 1 g Esculin sesquihydrate | Carl Roth GmbH + Co. KG, Germany |
| 1 g Ammonium ferric citrate | Sigma-Aldrich®, United States |
| 1000 ml dH2O |  |
| LB-Agar-CMC | 40 g LB-Agar (Luria/Miller) \*Dehydrated commercial culture media | Carl Roth GmbH + Co. KG, Germany |
| 10 g CMC | Carl Roth GmbH + Co. KG, Germany |
| 1000 ml dH2O |  |
| LB-Agar-Avicel | 40 g LB-Agar (Luria/Miller) \*Dehydrated commercial culture media | Carl Roth GmbH + Co. KG, Germany |
| 10 g Avicel PH-101 | Sigma-Aldrich®, United States |
| 1000 ml dH2O |  |
| CMC-Agar | 15 g Agar | Carl Roth GmbH + Co. KG, Germany |
| 10 g CMC | Carl Roth GmbH + Co. KG, Germany |
| 1000 ml dH2O |  |
| PASC-Agar | ≤ 0.2 g PASC | Carl Roth GmbH + Co. KG, Germany |
| 0.7 g Agar |  |
| 100 ml dH2O |  |
| PASA-Agar | ≤ 0.2 g PASA | Carl Roth GmbH + Co. KG, Germany |
| 1.4 g Agar |  |
| 100 ml dH2O |  |
| Cellulose overlay | 0.2 g cellulose | Carl Roth GmbH + Co. KG, Germany |
| 0.7 g Agar |  |
| 100 ml ml dH20 |  |

Table 25: Antibiotic supplements.

| Antibiotic | Stock solution | Final concentration | Manufacturer |
| --- | --- | --- | --- |
| Ampicillin | 100 mg/ml in dH2O | 100 µg/ml | Carl Roth® GmbH + Co. KG, Germany |
| Chloramphenicol | 35 mg/ml in ethanol (70 %) | 35 µg/ml | Merck Milipore, United States |
| Chloramphenicol | 5 mg/ml in ethanol (70 %) | 5 µg/ml | Merck Milipore, United States |
| Erythromycin | 1 mg/ml in ethanol (70 %) | 1 µg/ml | Sigma-Aldrich®, United States |
| Lincomycin | 25 mg/ml in dH2O | 25 µg/ml | Sigma-Aldrich®, United States |

---

### Strains

All strains used in this project are listed in [Tab.
26](#tab27)

cmr: chloramphenicol resistance, mlsr: erythromycin/ lincomycin resistance, ampr:
ampicillin resistance, neor: neomycin

Table 26: Strains.

| Strain | Description | Reference |
| --- | --- | --- |
| *E. coli* | | |
| TME4423 | DH10β pBS1C-P*cotYZ*-sfGFP-cotY, *ampr* | Laboratory collection |
| *E. coli*  | Laboratory collection | |
| *E. coli* DH10β | *ФlacZ ΔM15 recA1 endA1 gyrA96 thi-1 hsdR17 (rK-,mK+) supE44 relA1 deoR Δ(lacZYA-arg F)* U169 | Laboratory collection |
| *B. subtilis* | | |
| W168 | laboratory wildtype strain, *B. subtilis trpC2* | Laboratory collection |
| WB800N | *B. subtilis nprE aprE epr bpr mpr::ble nprB::bsr vpr wprA::hyg cm::neo; neor (cross-resistance to kanamycin)* | [MoBiTec GmbH](https://www.mobitec.com/products/vector-systems/bacillus-expression-systems/pbs022/bacillus-subtilis-strain-wb800n-for-secretion-vectors) |
| 3A38 | *B. subtilis* NCIB 3610 comIQ12L (= strain DK1042) | [BGSC](https://bgsc.org/getdetail.php?bgscid=3A38) |
| WB600 | *B. subtilis trpC2 ΔnprE ΔaprA Δepr Δbpf Δmpr ΔmprB* | [Wu *et al.* 1991](https://journals.asm.org/doi/10.1128/jb.173.16.4952-4958.1991) |
| BKE18130 | *B. subtilis* W168 trpC2 *ΔeglS::erm, mlsr* | [Koo *et al.* 2017](https://www.sciencedirect.com/science/article/pii/S2405471216304471?via%3Dihub) |
| BKE03410 | *B. subtilis* W168 *∆bglC::erm, mlsr* | [Koo *et al.* 2017](https://www.sciencedirect.com/science/article/pii/S2405471216304471?via%3Dihub) |
| BKE40110 | *B. subtilis* W168 trpC2 *∆bglA::erm, mlsr* | [Koo *et al.* 2017](https://www.sciencedirect.com/science/article/pii/S2405471216304471?via%3Dihub) |
| BKE39070 | *B. subtilis* W168 trpC2 *∆bglS::erm, mlsr* | [Koo *et al.* 2017](https://www.sciencedirect.com/science/article/pii/S2405471216304471?via%3Dihub) |
| BKE38560 | *B. subtilis* W168 trpC2 *∆licH::erm, mlsr* | [Koo *et al.* 2017](https://www.sciencedirect.com/science/article/pii/S2405471216304471?via%3Dihub) |
| BKE39260 | *B. subtilis* W168 trpC2 *∆bglH::erm, mlsr* | [Koo *et al.* 2017](https://www.sciencedirect.com/science/article/pii/S2405471216304471?via%3Dihub) |

---

### Vectors

All vectors used in this project are listed in [Tab.
27](#tab28).

Table 27: Vectors.

| Vector | Description | Reference |
| --- | --- | --- |
| pSB1C | pDG1662 derivative; empty, *amyE'…'amyE* integration in **B. subtilis*, catr, blar*, ColE1 | [Radeck *et al.* 2013](https://jbioleng.biomedcentral.com/articles/10.1186/1754-1611-7-29) |
| pBS1C3 | *ori* pMB1, cmr, rfp-cassette in BioBrick MCS | iGEM |
| pBS2E-*xylR*-P*xylA*-*xylR* | pBS2E derivative, xylose inducible Pxyl (with regulator XylR) in front of mcs; BioBrick compatible, rfp in mcs; blar, mlsr, integrates in lacA | [Popp *et al.* 2017](https://www.nature.com/articles/s41598-017-15107-z) |
| pBS0E-xylR-P*xylA* | pBS0E derivative; xylose inducible Pxyl (with regulator XylR) in front of mcs; BioBrick compatible, rfp in mcs; blar, mlsr, *ori1030*, ColE1 | [Popp *et al.* 2017](https://www.nature.com/articles/s41598-017-15107-z) |
| pDG1726 | pSB119, *aad9* | [Guerout-Fleury *et al.* 1995](https://www.sciencedirect.com/science/article/pii/0378111995006524) |
| pDG1513 | pMTL22, *tetr* | [Guerout-Fleury *et al.* 1995](https://www.sciencedirect.com/science/article/pii/0378111995006524) |

---

## References

---

* MoBiTec GmbH:
  https://www.mobitec.com/products/vector-systems/bacillus-expression-systems/pbs022/bacillus-subtilis-strain-wb800n-for-secretion-vectors,
  Accessed 26.09.2024, 17:10.
* BGSC: https://bgsc.org/getdetail.php?bgscid=3A38, Accessed 26.09.2024, 17:12.
* Wu, Xu-Chu, *et al.* "Engineering a *Bacillus subtilis* expression-secretion system
  with a strain
  deficient in six extracellular proteases." Journal of bacteriology 173.16 (1991): 4952-4958.
* Koo, Byoung-Mo *et al.* “Construction and Analysis of Two Genome-Scale Deletion Libraries
  for
  *Bacillus subtilis*.” Cell systems vol. 4,3 (2017): 291-305.e7.
  doi:10.1016/j.cels.2016.12.013
* Korotkova, O. G., Semenova, M. V., Morozova, V. V., Zorov, I. N., Sokolova, L. M., Bubnova, T. M.,
  Okunev, O. N., & Sinitsyn, A. P. (2009). Isolation and properties of fungal beta-glucosidases. Biochemistry.
  Biokhimiia, 74(5), 569–577. https://doi.org/10.1134/S0006297909050137
* McCarthy, J. K., Uzelac, A., Davis, D. F., & Eveleigh, D. E. (2004). Improved Catalytic Efficiency
  and Active Site Modification of 1,4-β-d-Glucan Glucohydrolase A from *Thermotoga neapolitana* by Directed Evolution.
  Journal of Biological Chemistry, 279(12), 11495–11502. https://doi.org/10.1074/JBC.M305642200
* Radeck, Jara *et al.* “The *Bacillus* BioBrick Box: generation and evaluation of
  essential
  genetic
  building blocks for standardized work with *Bacillus subtilis*.” Journal of biological
  engineering vol. 7,1 29. 2 Dec. 2013, doi:10.1186/1754-1611-7-29
* Popp, Philipp F *et al.* “The *Bacillus* BioBrick Box 2.0: expanding the genetic
  toolbox for the
  standardized work with *Bacillus subtilis*.” Scientific reports vol. 7,1 15058. 8 Nov.
  2017,
  doi: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/bm050799c
* Guérout-Fleury, A M *et al.* “Antibiotic-resistance cassettes for *Bacillus
  subtilis*.” Gene vol.
  167,1-2 (1995): 335-6. doi:10.1016/0378-1119(95)00652-4
* 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

{% endautoescape %}
{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content2 %}

* .
* OUTLINE
* [Phase 1: Literature Research
  ![](https://static.igem.wiki/teams/5117/images-wiki/plus-circle.svg)](#submenu1)
  + [Selection on Enzyme Candidates](#href_result1)
* [Phase 2: Subcloning
  ![](https://static.igem.wiki/teams/5117/images-wiki/plus-circle.svg)](#submenu2)
  + [Subcloning of Enzyme Candidates into
    pSB1C3](#href_result2)
* [Phase 3: Induced Expression
  ![](https://static.igem.wiki/teams/5117/images-wiki/plus-circle.svg)](#submenu3)
  + [Cloning Enzyme Candidates into Inducible
    Expression Vectors](#href_result3)
  + [Activity Assays of Secreted and Intracellular
    Enzyme Candidates](#href_result4)
* [Phase 4: Spore Surface Display
  ![](https://static.igem.wiki/teams/5117/images-wiki/plus-circle.svg)](#submenu4)
  + [Cloning Enzyme Candidates fused to Anchor
    Protein into pBS1C](#href_result5)
  + [Activity Assays of Enzymes Immobilized on
    *B. subtilis* Spores](#href_result6)
* [Conclusion
  ![](https://static.igem.wiki/teams/5117/images-wiki/plus-circle.svg)](#submenu5)
  + [Project Summary](#href_result7)
  + [Project Achievements](#href_result8)
* [Outlook
  ![](https://static.igem.wiki/teams/5117/images-wiki/plus-circle.svg)](#submenu6)
  + [Considerations for Replicating
    Experiments](#href_result9)
  + [Future Plans](#href_result10)
* [References](#href_references)

---

# **Results**

---

## Overview

The results of our project are divided into four phases: First, a comprehensive literature review in which
we examined scientific papers and consulted experts with varying expertise to refine our project idea and
find suitable enzymes. Second, the subcloning of enzyme candidates as an intermediate step to generate template
plasmids for use in following project phases.

Third, results of our reference approach, induced expression, are explained in which enzyme candidates were
cloned into inducible expression vectors and activity assays were carried out to identify enzymes functional
in the host *Bacillus subtilis*. Fourth, and finally, promising enzymes were chosen for our final spore
surface
display strategy and analyzed by activity assays.

![](https://static.igem.wiki/teams/5117/images-wiki/results/0-results-projectphases-new.webp)

---

## Phase 1: Literature Research

### Selection of Enzyme Candidates

As soon as our team decided on the project topic “ReFiBa – Enzyme-Based Recycling of Textile Fibers Using
*Bacillus subtilis*”, we reviewed literature, looked at the work of previous iGEM teams and consulted local
experts,
all of which shaped our project (see [Engineering](https://2024.igem.wiki/tu-dresden/engineering) and
[Human Practices](https://2024.igem.wiki/tu-dresden/human-practices) page).

Our project is divided into two main strategies:

1) Induced expression as a reference approach, in which we aimed to find active cellulases and PETases
for working with *B. subtilis* as an expression host, and

2) Spore surface display as a final strategy, in which chosen enzymes are immobilized on the
surface of *B. subtilis* spores.

To begin, we needed to determine enzyme candidates capable of degrading cellulose and PET. All details and
information, which influenced the selection of enzyme candidates, can be reviewed on the [Engineering](https://2024.igem.wiki/tu-dresden/engineering) page.
Due to the phylogenetic status of *B. subtilis* as a Firmicute, cellulase candidates from this
phylum Firmicutes
(Bacillota) were ultimately chosen and thought to function well in the organism. The PETase candidate had
already been codon optimized for *Bacillus* (Xi *et al.*, 2021). The following table (Tab. 1) displays
all ten
enzyme candidates, the species in which they were found and our abbreviation (which will be used from now on).

**Table 1: Enzyme candidates for cellulose and PET
degradation.**

| Enzyme | Candidate | Species | Abbreviation |
| --- | --- | --- | --- |
| **Endoglucanase (EC 3.2.1.4)** | EgIS EgIA CeIA CeIG | *Bacillus subtilis* *Bacillus pumilus* *Acetivibrio thermocellus* *Acetivibrio thermocellus* | BsEglS BpEglA AtCelA AtCelG |
| **Exoglucanase (EC 3.2.1.176)** | CeIO CeIS | *Acetivibrio thermocellus* *Acetivibrio thermocellus* | AtCelO AtCelS |
| **β-Glucosidase (EC 3.2.1.21)** | BglA BglB BglA | *Bacillus halodurans* *Paenibacillus polymyxa* *Acetivibrio thermocellus* | BhBglA PpBglB AtBglA |
| **PETase (EC 3.1.1.101)** | PETase | *Bacterium HR29* | BhrPET |

Thereby, we achieved our first milestone:

**MILESTONE 1:**

**Decision on enzyme candidates for degradation of cellulose and PET**

**Endoglucanase, Exoglucanase , β-Glucosidase, PETase**

---

## Phase 2: Subcloning

### Subcloning of Enzyme Candidates into pSB1C3

After choosing ten enzyme candidates, we designed biological parts functioning as translational units containing
the ribosome
binding site for *B. subtilis* as well as the gene of interest. The parts were flanked by the BioBrick
prefix and suffix sequences
allowing for BioBrick standard assembly and restriction-ligation-cloning. All information on construct design
can
be found on the [Engineering](https://2024.igem.wiki/tu-dresden/engineering) page.

In order to create a template from which these parts could be amplified, we aimed to subclone the parts into a
small vector pSB1C3.
For that purpose, we isolated the plasmid from *E. coli* DH10β provided by the laboratory collection of
Prof. Thorsten Mascher, yielding
a DNA concentration of 431.0 ng/µl. Afterwards, we performed a Backbone PCR with pSB1C3 (Fig. 1) and
subsequently digested the amplified
vector backbone and the ordered parts with *Eco*RI and *Pst*I, which were purified via the
HiYield® PCR Clean-up/Gel
Extraction Kit (see [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page for detailed
protocols).

![](https://static.igem.wiki/teams/5117/images-wiki/results/1-backbone-psb1c3.webp)

**Figure 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](https://2024.igem.wiki/tu-dresden/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 plasmids were transformed into chemically competent *E. coli* DH10β cells.
Transformants were selected by
chloramphenicol resistance (35 μg/ml chloramphenicol) encoded on the pSB1C3 backbone (Fig. 2). 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 constructs, white colonies were tested for the presence of the correct insert by
Colony PCR
and agarose gel electrophoresis
 (Fig. 3).

![](https://static.igem.wiki/teams/5117/images-wiki/results/2-e-colitrafo-psb1c3-bhbgla.webp)

**Figure 2: *E. coli* DH10β transformants on LB + chloramphenicol plates.**

Negative control (NC) containing no DNA showed no growth at all. Positive control (PC) containing pSB1C3
resulted in a
pink bacterial lawn. Transformation with pSB1C3-BhBglA, for example, led to the growth of 35 white colonies

(colonies marked in green were chosen for Colony PCR).

![](https://static.igem.wiki/teams/5117/images-wiki/results/3-e-coli-colonypcr-psb1c3-bhbgla.webp)

**Figure 3: Agarose gel electrophoresis: Insert amplification of pSB1C3
backbone by Colony PCR of transformed *E. coli* DH10β cells, demonstrated with plasmid
pSB1C3-BhBglA.**

Oligonucleotides for amplification can be found on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page. Numbers 1-6 correspond to chosen
colonies. The correct PCR product has a size of 1673 bp. The negative control displayed no band, but was
loaded onto another gel and is therefore not shown here. 1 kb Plus DNA Ladder (NEB) served as marker (M).
Colony 6 resulted in the highest plasmid concentration and was subsequently verified by sequencing revealing
the correct insert sequence. Results for all parts are documented on the respective Parts Registry pages
(links can be found on the [Contribution](https://2024.igem.wiki/tu-dresden/contribution) page).

Colonies with a band the correct size of the insert were chosen for plasmid isolation according to the
HiYield® Plasmid Mini DNA Kit. Finally, ten plasmids each containing one of our ten enzyme candidates
were verified via sequencing by Microsynth Seqlab GmbH. These plasmids (Tab. 2) served as templates for
PCR amplification of parts required for the next phases of our project.

**Tab. 2: Generated plasmid templates and *E. coli* DH10β strains containing genes of interest.**

| Construct | DNA Concentration [ng/µl] | Plasmid No. | *E. coli* No. |
| --- | --- | --- | --- |
| **pSB1C3-BsEglS** | 297.6 | P01 | Ec01 |
| **pSB1C3-BpEglA** | 233.3 | P02 | Ec02 |
| **pSB1C3-AtCelA** | 140.3 | P03 | Ec03 |
| **pSB1C3-AtCelG** | 250.0 | P04 | Ec04 |
| **pSB1C3-AtCelO** | 322.7 | P05 | Ec05 |
| **pSB1C3-AtCelS** | 384.4 | P06 | Ec06 |
| **pSB1C3-BhBglA** | 252.7 | P07 | Ec07 |
| **pSB1C3-PpBglB** | 262.4 | P08 | Ec08 |
| **pSB1C3-AtBglA** | 277.5 | P09 | Ec09 |
| **pSB1C3-BhrPET** | 112.8 | P10 | Ec10 |

---

## Phase 3: Induced Expression

### Cloning Enzyme Candidates into Inducible Expression Vectors

After subcloning our biological parts into the small vector pSB1C3, we started project phase 3 focusing
on testing the functionality and activity of chosen enzyme candidates. For that purpose, the parts were
cloned into xylose-inducible expression vectors to overexpress our target genes and both replicative
(pBS0E-*xylR*-P*xylA*) and integrative (pBS2E-*xylR*-P*xylA*) vectors
were used (Popp *et al.*, 2017). Details and
vector maps are documented on the [Engineering](https://2024.igem.wiki/tu-dresden/engineering) page.

The vectors were isolated from *E. coli* DH10β provided by the laboratory collection of Prof. Thorsten
Mascher,
resulting in DNA concentrations of 67.5 ng/µl pBS0E-*xylR*-P*xylA* and 165.9 ng/µl
pBS2E-*xylR*-P*xylA*. These vectors
were digested with *Eco*RI and *Pst*I (Fig. 4) and purified via gel extraction using the
HiYield® PCR Clean-up/Gel
Extraction Kit.

![](https://static.igem.wiki/teams/5117/images-wiki/results/4-res-pbs0ex-pbs2ex.webp)

**Figure 4: Agarose gel electrophoresis: Restriction (Res) of expression
vectors pBS0E-*xylR*-P*xylA* (pBS0EX) and pBS2E-*xylR*-P*xylA*
(pBS2EX) with *Eco*RI and *Pst*I.**

The digested plasmid backbones have sizes of 8114 bp and 7758 bp, respectively. Strong bands at approximately
9000 bp could represent undigested plasmids as well as digested ones due to high size inaccuracy of large
bands.
Weak bands at 1102 bp represent the RFP insert being cut out of the vector. Large bands were purified by gel
extraction and resulted in 18.5 ng/µl and 26.9 ng/µl DNA for the digested vectors pBS0EX and pBS2EX,
respectively.
1 kb Plus DNA Ladder (NEB) served as marker (M).

Biological parts previously digested with *Eco*RI and *Pst*I in the subcloning phase were ligated into
the
digested expression vectors. In case there was not enough restriction product left, parts were amplified
via PCR (Fig. 5) using the plasmids from project phase 1 as a template (e.g. pSB1C3-BhBglA) and subsequently
digested and purified via PCR clean up (see [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page for detailed protocols).

![](https://static.igem.wiki/teams/5117/images-wiki/results/5-pcr-bhbgla.webp)

**Figure 5: Agarose gel electrophoresis: PCR of part BhBglA.**

Oligonucleotides for amplification can be found on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page. The correct PCR product
has a size of 1410 bp. The larger band probably represents the plasmid pSB1C3-BhBglA used as template.
1 kb Plus DNA Ladder (NEB) served as marker (M). BhBglA was purified by gel extraction resulting in
a DNA concentration of 174.3 ng/µl. Results for all parts are documented on the respective Parts
Registry pages (links can be found on the [Contribution](https://2024.igem.wiki/tu-dresden/contribution) page).

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. However,
no growth was observed for some constructs (Fig. 6 A), which may be due to very low transformation efficiency.

For those constructs, either the entire vector or the amplified vector backbone was digested and purified
by PCR clean up instead of gel extraction (DNA concentrations of digested
pBS0E-*xylR*-P*xylA*: 20.8 ng/µl,
25.9 ng/µl; digested pBS2E-*xylR*-P*xylA*: 24.7 ng/µl, 26.0 ng/µl). This approach finally
led to successful
transformation (Fig. 6 B). Nevertheless, the original vector insert containing RFP was not removed.
Colonies with re-ligated plasmids, however, appear pink on the plate due to the RFP insert and can therefore
be distinguished from transformants with the target insert.

![](https://static.igem.wiki/teams/5117/images-wiki/results/6-e-coli-trafo-pbs0ex-pbs2ex-bhbgla.webp)

**Figure 6: *E. coli* DH10β transformants on LB + ampicillin plates,
based
on vector purification by gel extraction (A) or by PCR clean up (B).**

**A:** Negative control (NC) containing no DNA showed no growth at all. Positive control (PC) containing
pBS0E-*xylR*-P*xylA* (pBS0EX) resulted in a pink bacterial lawn. Transformation is
demonstrated with plasmid
pBS0EX-BhBglA and led to the growth of only 2 white colonies, indicating low transformation efficiency.

**B:** Negative control (NC) containing no DNA showed no growth at all. Positive control (PC) containing
pBS2E-*xylR*-P*xylA* (pBS2EX) resulted in ≈ 400 pink colonies. Re-ligation controls
containing the digested
vectors pBS0E-*xylR*-PxylA (RC1) and pBS2E-xylR-PxylA (RC2) led to growth of ≈ 300 pink
and 17 white colonies
(RC1), and ≈ 400 pink and 28 white colonies (RC2), respectively. The transformation of pBS0EX-BhBglA
led to the growth of ≈ 200 colonies (ratio 1:1 pink and white) and that of pBS2EX-BhBglA to ≈ 300
colonies (ratio 1:1 pink and white). These results suggest that vector purification by PCR clean up
result in higher transformation efficiency. However, white colonies are not necessarily correct as
the re-ligation controls showed few white colonies as well (barely visible in the photos).

White colonies transformed with the expression plasmids were analyzed by Colony PCR and agarose gel
electrophoresis (Fig. 7). Colonies with a band of the correct size of the insert were chosen for plasmid
isolation. Finally, ten replicative and ten integrative expression plasmids containing one of our
ten enzyme candidates each were verified by sequencing.

![](https://static.igem.wiki/teams/5117/images-wiki/results/7-e-coli-colonypcr-pbs0ex-pbs2ex-bhbgla.webp)

**Figure 7: Agarose gel electrophoresis: Insert amplification of (A)
pBS0E-*xylR*-P*xylA* (pBS0EX) and (B) pBS2E-*xylR*-P*xylA*
(pBS2EX) by Colony PCR of transformed *E. coli*
DH10β cells, demonstrated with part BhBglA.**

Oligonucleotides for amplification can be found on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page. Numbers 1-6 correspond
to chosen colonies. Correct PCR products have a size of 1561 bp for pBS0EX-BhBglA (A) and 1762 bp
for pBS2EX-BhBglA (B). Negative controls (NC) displayed no band. Whereas the NC for pBS0EX is
shown here, the NC for pBS2EX was loaded onto another gel and is therefore not depicted. 1 kb
Plus DNA Ladder (NEB) served as marker (M). Colony 2 of pBS0EX-BhBglA and colony 4 of pBS2EX-BhBglA
were verified by sequencing and contained the correct insert sequence. Results for all parts
are documented on the respective Parts Registry pages (links can be found on the [Contribution](https://2024.igem.wiki/tu-dresden/contribution) page).

Ultimately, these expression plasmids were transformed into our target host *B. subtilis*. Since our
reference strategy focused on the secretory expression of target enzymes (except for β-glucosidases),
we chose WB800N 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).

Since we discovered background activity in initial quality activity assays (see [Activity
assays of
secreted and intracellular enzyme candidates](#href_result4)), we aimed to generate *B. subtilis* WB800N deletion
strains (Δ*eglS*, Δ*bglH*) to remove the background signal caused by the native endoglucanase EglS and
β-glucosidase BglH. To achieve this, we sought to replace the gene with a resistance cassette to generate knockouts.

We first amplified the spectinomycin resistance cassette and up- and downstream sequences of the gene
to be knocked out by PCR before joining these fragments in an Overlap PCR (see [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page for
detailed protocols). Here the first difficulties arose, as the ratio and concentration of the different
fragments to each other are crucial for efficient amplification of the target product. Several ratios
and concentrations were tested (see [Experiments](https://2024.igem.wiki/tu-dresden/experiments)),
with 10 ng each of up and down fragments and 30 ng
of spectinomycin cassette being the best tested option (Fig. 8).

![](https://static.igem.wiki/teams/5117/images-wiki/results/8-overlappcr-knockout-egls-bglh-new.webp)

**Figure 8: Agarose gel electrophoresis: Amplification of *eglS* and
*bglH*
knockout constructs by Overlap PCR using a spectinomycin resistance cassette (*spec*).**

Oligonucleotides for amplification can be found on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page. Correct PCR products have
a size of 3264 bp for the *eglS* knockout construct and 3252 bp for the *bglH* knockout construct.
1 kb Plus DNA Ladder (NEB) served as marker (M)

However, transformation of the constructs into *B. subtilis* WB800N failed several times, resulting in
no growth or few false colonies. Even an attempt to use a different resistance cassette (tetracycline)
resulted in problems with the efficiency of construct amplification and no transformation success
(iteration details can be found on [Engineering](https://2024.igem.wiki/tu-dresden/engineering)
page).

After multiple failed attempts to generate the deletion strains, we decided to discard this idea and
to transform expression plasmids directly into WB800N. Being aware that there is some background
signal, this strain will be included as a control in all activity assays. The transformants were
selected by MLS resistance (1 µg/ml erythromycin and 25 µg/ml lincomycin) encoded on the vector backbones (Fig.
9).

Since the *Bacillus* transformation did not work for all plasmids, the transformation was repeated
with earlier addition of DNA to growing WB800N cells (at OD600 ≈ 0.7 instead of 1.1) 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](https://2024.igem.wiki/tu-dresden/experiments) page).

![](https://static.igem.wiki/teams/5117/images-wiki/results/9-b-subtilis-trafo-pbs0ex-pbs2ex-bhbgla.webp)

**Figure 9: *B. subtilis* WB800N transformants on LB + MLS plates.**

Negative control (NC) containing no DNA showed to no growth. Transformation with
pBS0E-*xylR*-P*xylA*-BhBglA
(pBS0EX-BhBglA), for example, led to the growth of ≈ 80 white colonies. Transformation with
pBS2E-*xylR*-PxylA-BhBglA (pBS2EX-BhBglA), which was linearized by restriction with
*Bsa*I prior to transformation, resulted in ≈ 50 white colonies. Colonies
marked in green were chosen for Colony PCR.

Finally, all remaining 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. 10). Two
colonies
each with the correct insert size were chosen for cryo-conservation, serving as biological duplicates.

![](https://static.igem.wiki/teams/5117/images-wiki/results/10-b-subtilis-colonypcr-pbs0ex-pbs2ex-bhbgla.webp)

**Figure 10: Agarose gel electrophoresis: Insert amplification of (A)
pBS0E-*xylR*-P*xylA* (pBS0EX) and (B) pBS2E-*xylR*-P*xylA*
(pBS2EX) by Colony PCR of transformed *B. subtilis*
WB800N cells, demonstrated with part BhBglA.**

Oligonucleotides for amplification can be found on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page. Numbers 1-4 correspond
to chosen colonies. 1 kb Plus DNA Ladder (NEB) served as marker (M). Results for all parts are
documented on the respective Parts Registry pages (links can be found on the [Contribution](https://2024.igem.wiki/tu-dresden/contribution) page).

**A:** Primer pair 1 was used for the amplification of a small fragment including BhBglA only (1410 bp).
Primer pair 2 was used for amplification of a large fragment including
*xylR*-P*xylA*-BhBglA (3041 bp).
Colonies 2 and 4 of pBS0EX-BhBglA were correct and chosen for cryo-conservation.

**B:** Primer pair 3 was used to check downstream integration by amplification of a fragment
including BhBglA and *'lacA* (1948 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).
As only colonies 1 and 4 displayed a correct band after PCR using primer pair 3, these were tested
using primer pair 4. Colony PCR for colony 1 was repeated after no visible band at first attempt.
Colonies 1 and 4 of pBS2EX-BhBglA were correct and chosen for cryo-conservation.

**C:** Negative controls of all primer pairs (NC 1-4) displayed no bands.

In the end, we successfully generated all 20 expression plasmids, both integrative
and replicative, as well as their corresponding *E. coli* and *B. subtilis* strains
(Tab. 3, Tab. 4), the latter of which were used for subsequent activity tests.

Tab. 3: Replicative expression plasmids, *E. coli*
DH10β and *B. subtilis* WB800N strains.

| Construct | DNA Concentration [ng/µl] | Plasmid No. | *E. coli* No. | *B. subtilis* No. |
| --- | --- | --- | --- | --- |
| **pBS0E-*xylR*-P*xylA*-BsEglS** | 601.3 | P11 | Ec11 | Bs01 |
| **pBS0E-*xylR*-P*xylA*-BpEglA** | 427.9 | P12 | Ec12 | Bs02 |
| **pBS0E-*xylR*-P*xylA*-AtCelA** | 388.6 | P13 | Ec13 | Bs03 |
| **pBS0E-*xylR*-P*xylA*-AtCelG** | 600.7 | P14 | Ec14 | Bs04 |
| **pBS0E-*xylR*-P*xylA*-AtCelO** | 266.2 | P15 | Ec15 | Bs05 |
| **pBS0E-*xylR*-P*xylA*-AtCelS** | 471.1 | P16 | Ec16 | Bs06 |
| **pBS0E-*xylR*-P*xylA*-BhBglA** | 405.8 | P17 | Ec17 | Bs07 |
| **pBS0E-*xylR*-P*xylA*-PpBglB** | 504.2 | P18 | Ec18 | Bs08 |
| **pBS0E-*xylR*-P*xylA*-AtBglA** | 601.6 | P19 | Ec19 | Bs09 |
| **pBS0E-*xylR*-P*xylA*-BhrPET** | 428.2 | P20 | Ec20 | Bs10 |

Tab. 4: Integrative expression plasmids, *E. coli*
DH10β and *B. subtilis* WB800N strains.

| Construct | DNA Concentration [ng/µl] | Plasmid No. | *E. coli* No. | *B. subtilis* No. |
| --- | --- | --- | --- | --- |
| **pBS2E-*xylR*-P*xylA*-BsEglS** | 93.6 | P21 | Ec21 | Bs11 |
| **pBS2E-*xylR*-P*xylA*-BpEglA** | 138.1 | P22 | Ec22 | Bs12 |
| **pBS2E-*xylR*-P*xylA*-AtCelA** | 148.2 | P23 | Ec23 | Bs13 |
| **pBS2E-*xylR*-P*xylA*-AtCelG** | 140.6 | P24 | Ec24 | Bs14 |
| **pBS2E-*xylR*-P*xylA*-AtCelO** | 98.5 | P25 | Ec25 | Bs15 |
| **pBS2E-*xylR*-P*xylA*-AtCelS** | 192.4 | P26 | Ec26 | Bs16 |
| **pBS2E-*xylR*-P*xylA*-BhBglA** | 147.8 | P27 | Ec27 | Bs17 |
| **pBS2E-*xylR*-P*xylA*-PpBglB** | 79.3 | P28 | Ec28 | Bs18 |
| **pBS2E-*xylR*-P*xylA*-AtBglA** | 92.2 | P29 | Ec29 | Bs19 |
| **pBS2E-*xylR*-P*xylA*-BhrPET** | 124.6 | P30 | Ec30 | Bs20 |

Additionally, we noticed that our replicative vector pBS0E-*xylR*-P*xylA*
does not contain a terminator after the insertion site of our constructs.
We therefore cloned all replicative plasmids with an additional terminator
as a backup option in case there were any expression problems.

For this, the bidirectional terminator B0014 was amplified from a plasmid
template provided by the laboratory collection, whereby the BioBrick prefix
and suffix were added by oligonucleotides, followed by purification via gel
extraction. All generated replicative plasmids (Tab. 3) were digested with *Spe*I
and *Pst*I. The terminator was digested with *Xba*I and *Pst*I. Both the plasmids and
the terminator were purified via PCR clean up. The terminator was then ligated
into each replicative plasmid due to complementary sticky ends of *Xba*I/*Spe*I
and *Pst*I/*Pst*I (see [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page
for detailed protocols).

The replicative plasmids containing the terminator were transformed into
chemically competent *E. coli* DH10β and transformants were selected by ampicillin
(100 μg/ml ampicillin). White colonies were checked for the presence of the
terminator by colony PCR and agarose gel electrophoresis (Fig. 11). Colonies
with a band of the correct size were selected for plasmid isolation using
the HiYield® Plasmid Mini DNA Kit. The insertion of the terminator into all
replicative plasmids was verified via sequencing by Microsynth Seqlab GmbH.

![](https://static.igem.wiki/teams/5117/images-wiki/results/11-colonypcr-pbs0ex-bhbgla-term.webp)

**Figure 11: Agarose gel electrophoresis: Verification of terminator
insertion in pBS0E-*xylR*-P*xylA* (pBS0EX) by Colony PCR of transformed
*E. coli* DH10β cells, demonstrated with part BhBglA.**

Oligonucleotides for amplification can be found on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page. Numbers 1-4 correspond
to chosen colonies. The correct PCR product has a size of 1664 bp. Colony PCR of the plasmid
without terminator served as a control (1561 bp). Negative control (NC) displayed no band.
1 kb Plus DNA Ladder (NEB) served as marker (M). Colony 2 was verified by sequencing and contained the correct
insert sequence.

However, these generated plasmids (Tab. 5) were not used further in our project,
since no expression problems occurred during testing of expression plasmids
without a terminator (see Activity assays of secreted and intracellular enzyme candidates).

**Tab. 5: Replicative expression plasmids with
additional B0014 terminator and respective *E. coli* DH10β strains.**

| Construct | DNA Concentration [ng/µl] | Plasmid No. | *E. coli* No. |
| --- | --- | --- | --- |
| **pBS0E-*xylR*-P*xylA*-BsEglS-B0014** | 563.4 | P31 | Ec31 |
| **pBS0E-*xylR*-P*xylA*-BpEglA-B0014** | 607.4 | P32 | Ec32 |
| **pBS0E-*xylR*-P*xylA*-AtCelA-B0014** | 439.1 | P33 | Ec33 |
| **pBS0E-*xylR*-P*xylA*-AtCelG-B0014** | 566.2 | P34 | Ec34 |
| **pBS0E-*xylR*-P*xylA*-AtCelO-B0014** | 364.9 | P35 | Ec35 |
| **pBS0E-*xylR*-P*xylA*-AtCelS-B0014** | 551.9 | P36 | Ec36 |
| **pBS0E-*xylR*-P*xylA*-BhBglA-B0014** | 539.5 | P37 | Ec37 |
| **pBS0E-*xylR*-P*xylA*-PpBglB-B0014** | 495.5 | P38 | Ec38 |
| **pBS0E-*xylR*-P*xylA*-AtBglA-B0014** | 604.2 | P39 | Ec39 |
| **pBS0E-*xylR*-P*xylA*-BhrPET-B0014** | 127.3 | P40 | Ec40 |

### Activity Assays of Secreted and Intracellular Enzyme Candidates

For the expression and testing of heterologously expressed enzymes in *B. subtilis*, we induced protein
production by adding 0.5 % xylose when cultures reached an OD600 of 0.5–0.6. After 24 hours, supernatants
were collected to test secreted enzymes (endoglucanases, exoglucanases, PETase), while intracellular
enzymes (β-glucosidases) were tested using liquid cultures and cell lysate. The enzyme activity of lysates
and supernatants was assessed, with additional samples analyzed by SDS-PAGE to confirm protein expression.
Detailed procedures are available on the [Experiments](https://2024.igem.wiki/tu-dresden/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 BhBglA, as "pBS0EX-BhBglA". Similarly, *B. subtilis* WB800N
transformed with the integrative plasmid containing the same enzyme will be referred to as "pBS2EX-BhBglA".

**Endoglucanases**

The functionality of the endoglucanases was investigated with a qualitative plate test using
1 % carboxymethyl cellulose (CMC) as substrate. First, the protocol was tested with a commercially
available cellulase mixture Accellerase 1500 (Genencor International, United States). After staining
the plates with congo red and subsequently destaining them with 1 M NaCl solution, a yellow halo was visible (as
shown in Fig. 12).

![](https://static.igem.wiki/teams/5117/images-wiki/results/1-endoglucanases.webp)

**Figure 12: Establishment of the qualitative assay for
determination of the endoglucanase activity.**

The enzyme mix was added into the well on LB-Agar-CMC plate and incubated for a day at 50 °C.

Mix Ac: Accellerase 1500, DF: dilution factor, NC: negative control.

Next, the background activity of different *B. subtilis* strains was tested. Overnight cultures
of all studied strains were prepared, adjusted to OD600 of 0.5, and 10 µl of each culture was
pipetted onto the LB-Agar-CMC plates. As positive control 10 μl of diluted 1:100 cellulase mix
Accellerase 1500 was added to the well made on the plate as it is described on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments)
page. All plates were incubated for 24 hours at 37 °C. Then, they were stained with congo red
solution and destained with 1 M NaCl to visualize halos (see [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page). The results are shown in Fig. 13.

![](https://static.igem.wiki/teams/5117/images-wiki/results/2-endoglucanases.webp)

**Figure 13: Screening of *B. subtilis* strains for background
endoglucanase activity.**

W168 is a laboratory wildtype strain, *B. subtilis* trpC2. In WB800N strain eight different proteases
are deleted. *bglH*, *bglS*, *eglS* and *licH* samples represent W168 strains with the
respective gene deletion
(see [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page). *E. coli* was applied
as negative control. The places where the colonies were
located before the staining process with congo red are marked with lines.

Mix Ac: Accellerase 1500, DF: dilution factor, NC: negative control.

Only with *B. subtilis* W168 trpC2 *ΔeglS::erm*, *mlsr* in which *eglS*, gene
encoding an endoglucanase,
was deleted, no background activity was detected. As earlier it was decided to produce enzymes
of interest in *B. subtilis* WB800N, an attempt was made to delete *eglS* in particularly this strain.
However, it failed, and the production of enzymes was performed in *B. subtilis* WB800N without further
deletions.

To determine CMCase activity of heterologous expressed endoglucanases (AtCelA, AtCelG, BsEglS, BpEglA) in *B.
subtilis*,
we first conducted a qualitative assay on CMC-Agar plates. For that, we first choose to test the strains
containing
the replicative pBS0E-*xylR*-P*xylA* vector assuming that the activity will be higher than
with the integrative
pBS2E-*xylR*-P*xylA* one.

After the expression of the proteins, we applied 15 µl of the supernatant to the wells in the center of the
CMC-Agar plates.
After 24 hours incubation at 50 °C, we stained plates with congo red method to visualize enzyme activity.
The results are shown in Fig. 14. The control strain WB800N and cultures without addition of inducer were used
as controls.
After induction with 0.5 % xylose, the activity was determined based on halo formation, which indicates CMC
degradation.
Negligible halos were observed on the plate of WB800N, indicating basal endoglucanase activity in the control
strain and
confirming the presence of the *eglS* gene in *B. subtilis*. Similarly, negligible halo formation was
observed in the
uninduced cultures, verifying that endoglucanase expression did not occur without induction, with only the basal
activity of EglS being present.

Clear halos were observed for BsEglS, BpEglA and AtCelA, indicating successful expression and activity of these
endoglucanases. The halo formation suggests that these enzymes are capable of degrading CMC. Since the activity
of BsEglS appeared to be comparable to that of BpEglA and higher than that of AtCelA, the latter was not
considered
for further experiments. Unfortunately, no halo formation was observed on plates with the supernatant of AtCelG,
indicating failed production in *B. subtilis* or very low endoglucanase activity under the conditions
tested.

![](https://static.igem.wiki/teams/5117/images-wiki/results/3-endoglucanases.webp)

**Figure 14: Qualitative CMCase activity assay of supernatant from induced
(with 0.5 % xylose) cultures of pBS0EX-AtCelA, pBS0EX-AtCelG, pBS0EX-BsEglS, and pBS0EX-BpEglA on CMC-agar
plates.**

To assess CMCase activity, 15 µL of the supernatant was applied to CMC-Agar plates. After incubation at
50 °C for 24 hours, plates were stained with congo red and destained with 1 M NaCl to visualize enzyme
activity. Clear halos around the wells indicate CMC degradation by the expressed endoglucanases. Uninduced
cultures and the strain WB800N were used as controls.

To decide whether BsEglS or BpEglA should be used for further immobilization on the spore surface, a
discontinuous
DNS assay was performed which is typically applied to estimate the amount of the reducing ends produced during
the
CMC degradation. At first, for this purpose a calibration curve was generated using calibration standards with
glucose concentrations ranging from 200 to 2000 μg/ml (as shown in Fig. 15).

![](https://static.igem.wiki/teams/5117/images-wiki/results/4-endoglucanases.webp)

**Figure 15: Glucose calibration curve for estimation of the produced reducing
ends with DNS assay.**

The glucose concentration in the calibration standards varied in the range from 200 to 2000 μg/ml.
The x-axis represents glucose concentration (μg/ml), and the y-axis shows the corresponding absorbance at
540 nm. A linear relationship between glucose concentration and absorbance is observed. This calibration
curve was used to calculate the concentration of the produced reducing ends in the reaction mixtures based on
their absorbance values.

For the DNS assay, supernatants diluted 1:14 were used, obtained from induced *B. subtilis* WB800N strains
containing the respective replicative pBS0E-*xylR*-P*xylA* plasmids. The reaction was
conducted with CMC diluted
in 50 mM phosphate buffer (pH 7) at 50 °C for different time intervals, ranging from 1 to 13 hours. DNS stop
solution was then applied as described on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page and absorbance was measured at 540
nm (see Fig. 16).
Controls included supernatants from *B. subtilis* WB800N, and supernatants from cultures without the
addition of a protein expression inducer.

![](https://static.igem.wiki/teams/5117/images-wiki/results/5-endoglucanases.webp)

**Figure 16: A discontinuous DNS assay to determine endoglucanase activity of
pBS0EX-BpEglA and pBS0EX-BsEglS.**

The assay was performed in 50 mM phosphate buffer pH 7 at 50 °C with 0.93 % CMC as substrate. The absorbance
was
measured at 540 nm. As samples served supernatants obtained from an untransformed *B. subtilis* WB800N
(WB800N),
*B. subtilis* WB800N transformed with a replicative plasmid containing the BsEglS or BpEglA genes
respectively
(pBS0EX-BsEglS, pBS0EX-BpEglA) as well as the supernatants from the respective strains after the induction of
protein expression with 0.5 % xylose (induced). The absorbance from a negative control containing only CMC in
a buffer was subtracted from the measured values to account for background signal. The experiment was conducted with a single biological replicate (N = 1).

Since *B. subtilis* naturally produces BsEglS, a notable background activity was detected after 13 hours of
reaction,
as previously shown with the qualitative CMC assay. However, this background activity did not interfere with the
assessment of recombinant protein activity. The amount of reducing ends produced in samples from uninduced
cultures
containing plasmids with BsEglS and BpEglA genes was comparable to the WB800N control, reaching 123.33 μg/ml and
113.33 μg/ml,
respectively, after 13 hours. Supernatants containing BpEglA led to the formation of 360.00 μg/ml reducing ends,
while BsEglS-containing supernatants showed the highest activity, correlating with 500.00 μg/ml of reducing ends
produced in 13 hours. Therefore, BsEglS was selected for further spore immobilization experiments.

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

**Exoglucanases**

The activity of exoglucanases was tested using a qualitative phosphoric acid swollen cellulose (PASC) assay and
the phosphoric acid swollen avicel (PASA) assay, which was developed as described on the [Engineering](https://2024.igem.wiki/tu-dresden/engineering) page. The
samples used included supernatants from untransformed *B. subtilis* WB800N and *B. subtilis* WB800N
transformed with
replicative pBS0E-*xylR*-P*xylA* vectors containing genes for AtCelS and AtCelO,
respectively. Unfortunately, no enzyme
activity was detected in any of the samples, as illustrated by the example of AtCelS (shown in Fig. 17). The
experiment
was repeated with supernatants from cultures in which protein expression was induced, not only at 37 °C as
before,
but also at 28 °C and 42 °C, however, still without success (data not shown). Because of this, we decided to
proceed
with both exoglucanase genes for protein immobilization on the spore surface.

![](https://static.igem.wiki/teams/5117/images-wiki/results/1-exoglucanases.webp)

**Figure 17: Qualitative assay for determination of exoglucanase activity on
PASA-Agar.**

Supernatants containing AtCelS, obtained from *B. subtilis* WB800N transformed with a replicative plasmid
containing the respective gene (pBS0EX-AtCelS), were added to wells on a plate with PASA agar. The assay was
used to evaluate protein expression and activity of AtCelS. A culture in which the inducer was not used served
as a control. For the induction of protein production, we used 0.5 % xylose (induced). After an incubation
period of 24 hours, a zone of clearance was expected, indicating exoglucanase activity.

**β-Glucosidases**

To examine the functioning of β-glucosidases, initially LB-Agar-Esculin plates were intended to be used.
For that purpose, 10 μl of cellulase mix Accellerase 1500 was added to the well made on the plate as it
is described on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page. Additionally,
different strains were tested to study the presence
of background activity. Overnight cultures of all generated strains were prepared, adjusted to OD600
of
0.5, and 10 µl of each culture was pipetted onto the plates, which were incubated for 24 hours at 37 °C
(as shown in Fig. 18). High background activity was observed in all studied strains. Nevertheless, it
was decided to test this qualitative assay for the recombinant proteins investigated in our project.

![](https://static.igem.wiki/teams/5117/images-wiki/results/1-glucosidases.webp)

**Figure 18: LB-Agar-Esculin plates to determine background activity of different *B. subtilis* strains.**

The cultures of both clones and the negative control (WB800N strain (upper panel) and *E. coli* (lower panel))
were grown overnight. Next day 10 µl of overnight culture with OD600 of 0.5, was added to the
plate and then incubated for 24 hours at 37 °C. The plates demonstrate the formation of black halos around
all cultures, including the negative control, indicating esculin degradation and suggesting
background activity from *B. subtilis* compared to *E. coli* (no black halo formation). No increase
in activity was observed on plates containing 0.5 % xylose as an inducer (induced).

To evaluate β-glucosidase activity, LB-Agar plates containing esculin were prepared, as described on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page.
Additionally, LB-Agar-Esculin plates, with and without 0.5 % xylose, were used to test whether our generated
intracellular
β-glucosidases could degrade esculin as an alternative substrate for cellobiose. Overnight cultures of all
generated strains
were grown, adjusted to OD600 of 0.5, and 10 µl of each culture was added to the plates, which were
incubated for 24 hours
at 37 °C (as shown in Fig. 19). We tested both *B. subtilis* clones that were generated in the cloning
procedure;
however, data for strains containing the integrative vector (pBS2E-*xylR*-P*xylA*) is not
shown, as no other results were obtained.

![](https://static.igem.wiki/teams/5117/images-wiki/results/2-glucosidases.webp)

**Figure 19: LB-Agar-Esculin plates to determine β-glucosidase activity of
both clones of pBS0EX-AtBglA, pBS0EX-BhBglA and pBS0EX-PpBglB.**

The cultures of both clones and the negative control (WB800N strain and *E. coli*) were grown overnight.
Next day 10 µl of
overnight culture with OD600 of 0.5, was added to the plate and then incubated for 24 hours at 37
°C. The plates
demonstrate the formation of black halos around all cultures, including the negative control, indicating
esculin degradation
and suggesting background activity from *B. subtilis* compared to *E. coli* (no black
halo formation). No increase
in activity was observed on plates containing 0.5 % xylose as an inducer (induced).

The assay revealed the formation of a black halo around all cultures, indicating esculin degradation. We hoped
for increased
activity on plates containing xylose, which was expected to induce expression of our enzymes, but no increase
was observed.
This may have been due to the background activity being too high to detect any differences. Unfortunately, even
the negative
control (WB800N) showed similar halo formation, suggesting high background activity from enzymes in *B.
subtilis* that are capable of
degrading esculin. Due to this background activity, the assay was discontinued.

Due to the high background activity observed in the previous assay (LB-Agar-Esculin plates), we decided to
quantify the
amount of glucose produced from the degradation of cellobiose because of the induced expression of our
glucosidases. To
access the intracellular enzymes to perform the reaction with cellobiose, we subsequently had to lyse the cells.

The glucose concentration in the reactions was determined using the Amplex™ Red Glucose/Glucose Oxidase Assay
Kit.
This assay involved preparing a working solution containing Amplex® Red reagent, horseradish
peroxidase (HRP),
and glucose oxidase, which was then mixed with glucose standards and samples (a more detailed protocol can be
found
on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page). A glucose calibration
curve was generated using standard concentrations ranging from 0 µM
to 150 µM, allowing for accurate quantification of glucose in subsequent reactions (as shown in Fig. 20).

![](https://static.igem.wiki/teams/5117/images-wiki/results/3-glucosidases.webp)

**Figure 20: Glucose standard calibration curve used for quantification of
glucose concentration
by degradation of cellobiose in glucose assays.**

The x-axis represents glucose concentration (µM), and
the y-axis shows the corresponding absorbance at 560 nm. A linear relationship between glucose concentration
and absorbance is observed.

To test the enzymatic activity of our glucosidases, we performed the expression protocol followed by the cell
lysis to obtain cell lysate, that was tested for β-glucosidases activity.

The lysates of pBS0EX-AtBglA, pBS0EX-BhBglA, and pBS0EX-PpBglB were incubated with 50 mM cellobiose at 50 °C for
up to 24 hours, followed by termination of the reaction. The glucose produced from cellobiose degradation was
measured using absorbance at 560 nm with the glucose assay to evaluate enzyme performance, as shown in Fig. 21.

![](https://static.igem.wiki/teams/5117/images-wiki/results/4-glucosidases.webp)

**Figure 21: Glucose concentration determination following cellobiose
degradation by intracellular β-glucosidases (lysate of pBS0EX-AtBglA, pBS0EX-BhBglA, pBS0EX-PpBglB) by using
the Amplex™ Red Glucose/Glucose Oxidase Assay Kit.**

The absorbance was measured at 560 nm, representing glucose production after degradation of 50 mM cellobiose
by
different β-glucosidases from lysate. WB800N was used as negative control. Dark purple bars represent
uninduced
(control) samples, light purple bars represent induced (0.5 % xylose) samples, and the green bar represents
the 50 mM cellobiose standard. The experiment was conducted with a single biological replicate (N = 1).

The results indicate that the induced expression (0.5 % xylose was used as an inducer) of pBS0EX-BhBglA produced
more glucose compared to its control and other enzymes, suggesting high enzymatic activity.
pBS0EX-AtBglA showed moderate activity, with slightly increased glucose production in the induced state compared
to
the control. Interestingly, pBS0EX-PpBglB exhibited higher absorbance in the control sample compared to the
induced
state, suggesting unexpected glucose production without induction.

The green bar, representing the cellobiose standard absorbance at 560 nm, suggests the presence of impurities in
the cellobiose, resulting in high background signal of the substrate. Furthermore, the lower absorbance values
of the enzyme samples compared to the substrate indicate potential interference from the lysate with the glucose
assay's coupled reaction, making the assay unsuitable for accurately testing lysate samples. The difference in
absorbance between cellobiose and pBS0EX-BhBglA corresponds to approx. 10 µM of glucose. Since the assay
involves
a 1:2 dilution of the sample, due to addition of working solution, the calculated glucose concentration should
be doubled. Therefore, the actual glucose concentration is approx. 20 µM. However, it should be noted that the
results might be inaccurate due to potential interference between the lysate and the assay.

Due to these unexpected results and potential interference from the lysate, we decided to use commercially
available *p*-nitrophenyl-β-D-glucopyranoside (pNPG) as an alternative substrate for analyzing the β-glucosidase
activity of our intracellular expressed enzymes.

The activity of β-glucosidase was determined by measuring the hydrolysis of pNPG, using the initial rate
of accumulation of the colored product, *p*-nitrophenol (pNP), following the method of Korotkova *et al.* (2009)
(see in [Experiments](https://2024.igem.wiki/tu-dresden/experiments)). For activity assessment, 20 µL
of β-glucosidase lysate (pBS0EX-BhBglA, pBS0EX-PpBglB,
pBS0EX-AtBglA, pBS2EX-BhBglA, pBS2EX-PpBglB, pBS2EX-AtBglA) was mixed with 180 µL of 5 mM pNPG substrate,
dissolved in 50 mM sodium phosphate buffer (pH 7.0) and incubated at 50 °C for 10 minutes. The reaction was
stopped by adding 100 µL of ice-cold 0.5 M Na2CO3, allowing for subsequent measurement
of pNP formation at 405 nm. The results are shown in Fig. 22. A negative control, without lysate, was included
to determine the background signal of the substrate, and the control values were subtracted from the obtained
results to reflect actual enzyme activity.

![](https://static.igem.wiki/teams/5117/images-wiki/results/5-glucosidases.webp)

**Figure 22: Determination of β-glucosidase activity of pBS0EX-AtBglA,
pBS0EX-BhBglA,
pBS0EX-PpBglB, pBS2EX-AtBglA, pBS2EX-BhBglA, pBS2EX-PpBglB by using pNPG as substrate.**

The absorbance was measured at 405 nm, representing pNP formation by degradation of pNPG by different
β-glucosidases. WB800N was used as negative control. Dark purple bars represent uninduced (control) samples,
light
purple bars represent induced (0.5 % xylose) samples. The experiment was conducted with a single biological replicate (N = 1).

The induced sample of pBS0EX-BhBglA shows a higher absorbance (approx. A405 = 2.5)
compared to the control,
indicating β-glucosidase activity. pBS0EX-AtBglA, pBS2EX-AtBglA, pBS2EX-BhBglA, pBS0EX-PpBglB, and
pBS2EX-PpBglB, show comparable
absorbance between induced and control samples, indicating minimal or no increase in activity upon induction.
This could be due to
insufficient enzyme yield during cell lysis, leading to reduced detectable activity. The negative control,
WB800N, shows similar
low absorbance for both conditions, confirming the absence of enzyme activity.

The results suggest that pBS0EX-BhBglA in the induced state exhibits higher enzyme activity, while
other samples
display almost no or minimal increase, suggesting lower or absent induction effects or absent enzyme activity
towards the substrate.

Based on the results shown in Fig. 21 and in Fig. 22, BhBglA demonstrated the highest activity, particularly
among the replicate vector
(pBS0EX strains), in both the glucose and pNPG assays, establishing it as the most promising candidate out of
three. Therefore,
BhBglA was selected for enzyme display on spores. Additionally, due to challenges associated with testing
lysate, PpBglB was also chosen
for spore display, supported by favorable findings from literature, despite its comparatively lower activity in
the assays.

**PETase**

To demonstrate the functionality of the produced BhrPET, a continuous *p*-nitrophenyl-acetate (pNPAc) assay was
conducted using
supernatants containing this enzyme. In this assay, pNPAc is degraded to acetate and pNP, which imparts a yellow
color to the solution.
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 BhrPET was included. Additionally,
supernatants obtained
from an untransformed *B. subtilis* WB800N were used as controls to check the ability of *B. subtilis*
to degrade pNPAc natively.
The reaction was performed in 50 mM phosphate buffer with pH 7 at 40 °C for 10 minutes (see [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page). The results are shown in Fig. 23.

The volume activity was calculated with extinction coefficient ε = 6.52 mM-1cm-1 (Kademi *et al.*, 2000). Notably, the influence of DMSO on the
pNP absorbance was neglected. In the future, to obtain more precise values for volume activity, the extinction
coefficient should be
determined experimentally for the exact conditions used. However, since the focus of our project was on the
enzyme immobilization on
spore surface of *B. subtilis*, no further assay optimizations were performed.

![](https://static.igem.wiki/teams/5117/images-wiki/results/1-petase.webp)

**Figure 23: Determination of PETase activity of supernatant of
pBS0EX-BhrPET.**

A continuous pNPAc assay was performed in 50 mM phosphate buffer pH 7 at 40 °C for 10 minutes to determine
PETase activity.
The absorbance was measured at 405 nm and the volume activity in U/ml was calculated. As samples served
supernatants obtained
from an untransformed *B. subtilis* WB800N (WB800N), *B. subtilis* WB800N transformed with a
replicative plasmid containing the BhrPET
gene (pBS0EX-BhrPET) as well as the supernatants from the respective strains after the induction of protein
expression with 0.5 % xylose (induced). The experiment was conducted with a single biological replicate (N = 1).

With pNPAc assay no background activity could be detected with supernatants obtained from *B.
subtilis* WB800N, while the activity of the
BhrPET-containing supernatant was successfully demonstrated, with an activity of 0.375 U/ml. Interestingly,
supernatants from the uninduced culture
showed slightly higher activity compared to the WB800N controls, indicating basal expression of the vector.

**SDS-PAGE**

To determine the molecular weight of the expressed proteins, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis
(SDS-PAGE) was performed using gels of varying concentrations (8 % for exoglucanases, 10 % for endoglucanases and
β-glucosidases, and 12 % for PETases).
Samples were prepared by mixing with SDS loading buffer and heating for protein denaturation. After loading the
samples and molecular weight marker
(PageRuler™ Plus Prestained Protein Ladder, 10 bis 250 kDa), gels were run at 100 V for approximately 1.5 hours.
The gels were stained with Coomassie
Blue and subsequently destained to visualize the proteins. Further details of the procedure can be found on the
[Experiments](https://2024.igem.wiki/tu-dresden/experiments) page.

To determine the molecular weight of heterologously expressed β-glucosidases, we expressed the strains
containing the pBS0E-*xylR*-P*xylA* vector (shown in Fig. 24).
As enzyme purification was not prioritized, and we applied cell lysate directly onto the gel, the presence of
multiple protein bands was expected. These bands represent
both the target enzymes and other cellular proteins. Consequently, the gel displays several protein bands, which
makes it challenging to identify the specific band
corresponding to the expressed β-glucosidase gene.

We expected to observe a band for AtBglA, BhBglA and for PpBglB between 51 – 53 kDa. However, due to the
overlapping bands from other cellular proteins, it remains difficult to
definitively pinpoint the specific bands for these expressed β-glucosidases.

![](https://static.igem.wiki/teams/5117/images-wiki/results/1-sdspage.webp)

**Figure 24: SDS-PAGE analysis of β-glucosidase lysates (pBS0EX-AtBglA,
pBS0EX-BhBglA, pBS0EX-PpBglB) on a 10 % gel.**

The figure shows lysate samples of induced (+) and uninduced (-) cultures. 4 µL of the PageRuler™ Plus
Prestained
Protein Ladder (10 to 250 kDa) (M) was loaded to estimate molecular weight, and the WB800N strain was used as
a
negative control. 45 µL of the lysate were mixed with 15 µL of 4X loading buffer and heated at 95 °C for 10
minutes
before loading 20 µL of each sample onto the gel. The gel was stained with Coomassie Blue and subsequently
destained
to visualize protein bands. The expected molecular weights for the target proteins are 51 – 53 kDa for AtBglA,
BhBglA and
PpBglB. Multiple protein bands are visible, indicating the presence of both target and non-target proteins in
the lysate
samples. Identification of the specific glucosidase bands is challenging due to overlapping signals.

We also analyzed the debris from β-glucosidase expression (as shown in Fig. 25). Like the results obtained from
the cell lysate, multiple bands
were observed on the gel, making it difficult to accurately identify the target protein bands based on molecular
weight. The presence of these
overlapping bands further complicates the identification of the specific β-glucosidase proteins.

![](https://static.igem.wiki/teams/5117/images-wiki/results/2-sdspage.webp)

**Figure 25: SDS-PAGE analysis of β-glucosidase debris (pBS0EX-AtBglA,
pBS0EX-BhBglA, pBS0EX-PpBglB) on a 10 % gel.**

The figure shows debris samples of induced (+) and uninduced (-) cultures. 4 µL of the PageRuler™ Plus
Prestained Protein Ladder (10 to 250 kDa) (M) was
loaded to estimate molecular weight, and the WB800N strain was used as a negative control. 45 µL of the debris
samples were mixed with 15 µL of 4X loading
buffer and heated at 95 °C for 10 minutes before loading 20 µL of each sample onto the gel. The gel was
stained with Coomassie Blue and subsequently destained
to visualize protein bands. The expected molecular weights for the target proteins are 51 – 53 kDa for AtBglA,
BhBglA and PpBglB. Multiple protein bands
are visible, indicating the presence of both target and non-target proteins in the debris samples.
Identification of the specific glucosidase bands is
challenging due to overlapping signals.

We also performed SDS-PAGE analysis for other heterologous expressed proteins, including endoglucanases (BpEglA
and BsEglS), exoglucanases (AtCelO and AtCelS),
and PETase (BhrPET), using the supernatant fraction (see [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page).

Since we did not detect any exoglucanase activity, we further analyzed the insoluble fraction (pellet)
alongside the supernatant, as shown in Fig. 26. Due to the lack of purification steps, it was difficult
to identify AtCelO and AtCelS. The expected molecular weights for the target proteins are 75 kDa for AtCelO
and 86 kDa for AtCelS. 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).

![](https://static.igem.wiki/teams/5117/images-wiki/results/3-sdspage.webp)

**Figure 26: SDS-PAGE analysis of exoglucanases (pBS0EX-AtCelO and pBS0EX-AtCelS)
supernatants and pellets on an 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 and 86 kDa for AtCelS. Multiple protein
bands are visible, indicating the presence of both target and non-target proteins in the soluble and insoluble fraction.

For endoglucanases, since activity was detected in our initial expression, we focused on BsEglS and BpEglA in
the SDS-PAGE analysis (shown in Fig. 27). Distinct
bands were observed in the induced culture lanes, corresponding to 55 kDa for BsEglS and 72 kDa for BpEglA,
verifying the expression of these proteins. However,
since BsEglS is originally a *B. subtilis* gene, slight bands were observed in all samples, confirming some
basal expression of this enzyme even without induction.

![](https://static.igem.wiki/teams/5117/images-wiki/results/4-sdspage.webp)

**Figure 27: SDS-PAGE analysis of endoglucanases supernatant (pBS0EX-BpEglA,
pBS0EX-BsEglS) on a 10 % gel.**

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

For our BhrPET we used both generated strains (pBS0EX-BhrPET and pBS2EX-BhrPET). The expected molecular weight
for the target protein, BhrPET,
is 25 kDa. Since no purification step was performed, multiple protein bands are visible, indicating the presence
of both target and non-target
proteins in the supernatant, as shown in Fig. 28. A very faint band is observed at the expected molecular
weight. However, to confirm the presence
of the target protein, further purification is necessary.

![](https://static.igem.wiki/teams/5117/images-wiki/results/5-sdspage.webp)

**Figure 28: SDS-PAGE analysis of PETases supernatant (pBS0EX-BhrPET,
pBS2EX-BhrPET) on a 12 % gel.**

The figure shows supernatant samples of induced (+) and uninduced (-) cultures. 4 µL of the PageRuler™ Plus
Prestained Protein Ladder
(10 to 250 kDa) (M) was loaded to estimate molecular weight, and the WB800N strain was used as a negative
control. 45 µL of
the supernatant were mixed with 15 µL of 4X loading buffer and heated at 95 °C for 10 minutes before loading 20 µL
of each sample
onto the gel. The gel was stained with Coomassie Blue and subsequently destained to visualize protein bands.
The expected
molecular weight for the target protein is 25 kDa for BhrPET. Multiple protein bands are visible, indicating
the presence
of both target and non-target proteins in the supernatant.

The SDS-PAGE analysis of β-glucosidases, endoglucanases, exoglucanases, and PETase consistently revealed
multiple protein bands,
indicating the presence of both target and non-target proteins. The absence of a purification step contributed
to the complexity
of the results, making it difficult to definitively identify the target protein bands, particularly when
multiple overlapping bands
were present. For some proteins, such as BhrPET, only faint bands were observed at the expected molecular
weight, whereas clear
bands at the correct size were only detected for BsEglS and BhEglA.

To address these challenges, purification using methods such as immobilized metal affinity chromatography (IMAC)
with the His-tag
of the proteins before SDS-PAGE could help reduce background proteins, allowing for clearer identification of
the target bands.
Additionally, Western blotting using specific antibodies could confirm the presence of the target proteins, even
when they are
present at low concentrations. Achieving higher protein expression levels could also improve the detection of
target proteins
in the gel. Alternatively, using a more sensitive staining method, such as silver staining instead of Coomassie
Blue, could enhance
the visibility of faint bands. Another possible solution would be to concentrate on the protein samples.
However, this would be
most effective after a purification step to avoid concentrating of non-target proteins as well.

In conclusion, our SDS-PAGE results indicate a need for further optimization of sample preparation and analysis
techniques to clearly
identify and confirm the presence of the target proteins. However, since the induced expression was primarily
intended to identify
candidates with desired enzymatic activity for spore display, we did not pursue additional solutions to improve
the purity of the
samples. Instead, we concluded that the observed enzyme activity was sufficient for our protein selection, and
therefore focused on
activity assays for further analysis.

**MILESTONE 2:**

**Decision on final enzymes for spore surface display**

**Endoglucanase: BsEglS**

**Exoglucanase: AtCelO, AtCelS**

**β-Glucosidase: BhBglA, PpBglB**

**PETase: BhrPET**

---

## Phase 4: Spore Surface Display

### Cloning Enzyme Candidates Fused to Anchor Protein Into pBS1C

After choosing enzymes for the spore surface display, we finally entered project phase 4 in which we
aimed to immobilize enzymes on *B. subtilis* spores. To express target genes only under sporulation, a
sporulation-dependent promoter P*cotYZ* was chosen. The target enzymes were fused to the
N-terminus of
the anchor protein CotY to anchor them on the spore surface.

We first tested N-terminal fusions, and due to limited time capacities, we were not able to test C-terminal
fusions as
well. Additionally, different linkers were analyzed: L1 – a short flexible linker, L2 – a long flexible linker
and
L3 – a rigid linker. All details about construct design are documented on the [Engineering](https://2024.igem.wiki/tu-dresden/engineering) page.

First, all biological parts including enzyme candidates with varying linkers (Fig. 29) as well as the promoter
P*cotYZ*,
the terminator B0014 and the anchor gene *cotY* (Fig. 30) were amplified by PCR and purified using the
HiYield® PCR
Clean-up/Gel Extraction Kit (see [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page
for detailed protocols). Plasmids generated in the subcloning
phase (Tab. 2) served as templates for PCR of enzyme candidates with the addition of linkers by
oligonucleotides.
Promoter and anchor genes were amplified from genomic DNA of *B. subtilis* W168 and the terminator from a
plasmid
provided by the laboratory collection.

![](https://static.igem.wiki/teams/5117/images-wiki/results/29-pcr-v2.webp)

**Figure 29: Agarose gel electrophoresis: PCR of parts for spore display
constructs demonstrated with part BhBglA including varying linkers (L1-L3).**

Oligonucleotides for amplification can be found on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page. Correct PCR products have a
size of 1394 bp, 1418 bp and 1421 bp, depending on the linker (L1-L3) added by oligonucleotides. 1 kb
Plus DNA Ladder (NEB) served as marker (M). PCR products were purified by gel extraction resulting in
DNA concentrations of 137.0 ng/µl (BhBglA-L1), 138.5 ng/µl (BhBglA-L2) and 162.9 ng/µl (BhBglA-L3),
respectively. Results for all parts are documented on the respective Parts Registry pages (links can
be found on the [Contribution](https://2024.igem.wiki/tu-dresden/contribution) page).

![](https://static.igem.wiki/teams/5117/images-wiki/results/13-pcr-pcotyz-coty-b0014.webp)

**Figure 30: Agarose gel electrophoresis: PCR of parts spore
display constructs: P*cotYZ*, *cotY* and B0014.**

Oligonucleotides for amplification can be found on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page. Amplification of
P*cotYZ*
results in a band of 238 bp, L1/L3-*cotY* displays a band of 514 bp, L2-*cotY* 544 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 and 97.3 ng/µl (L1/L3-*cotY*),
143.8 ng/µl (L2-*cotY*) and 52.2 ng/µl (B0014).

Parts were subsequently assembled via Overlap PCR by complementary overhangs which were
designed and added by oligonucleotides (Fig. 31). All Overlap PCR products of BsEglS, BhBglA,
PpBglB and BhrPET could be constructed. However, difficulties occurred during cloning of
larger enzyme candidates AtCelO and AtCelS, whose parts could not be assembled in time.
This was most likely due to faulty assembly caused by similarities between the overhangs and
sequences in the parts. Of the exoglucanases, only one Overlap PCR product was successfully
constructed: P*cotYZ*-AtCelO-L2-*cotY*-B0014.

![](https://static.igem.wiki/teams/5117/images-wiki/results/14-pcr-pcotyz-coty-b0014.webp)

**Figure 31: Agarose gel electrophoresis: Overlap PCR of spore display
constructs,
demonstrated with enzyme candidate BhBglA with varying linkers (L1-L3).**

Oligonucleotides for amplification can be found on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page. Correct PCR products have a size
of 2211 bp,
2265 bp and 2238 bp, depending on the linker (L1-L3). Other weak bands are probably caused by unspecific
binding of
oligonucleotides. 1 kb Plus DNA Ladder (NEB) served as marker (M). Overlap PCR products were purified by gel
extraction resulting in DNA concentrations of 155.8 ng/µL (BhBglA-L1), 135.4 ng/µL (BhBglA-L2) and 111.7 ng/µL
(BhBglA-L3). Results for all parts are documented on the respective Parts Registry pages (links can be found
on the [Contribution](https://2024.igem.wiki/tu-dresden/contribution) page).

Afterwards, Overlap PCR products were cloned into the vector backbone pBS1C enabling genome integration into the
*amyE* locus in
*B. subtilis* (Radeck *et al.*, 2013). For that purpose, inserts as well as pBS1C
were digested with *Eco*RI
and *Pst*I, whereas inserts were purified by PCR clean up and the vector by gel extraction (Fig. 32) using
the
HiYield® PCR Clean-up/Gel Extraction Kit (see [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page for detailed protocols).

![](https://static.igem.wiki/teams/5117/images-wiki/results/15-res-pbs1c.webp)

**Figure 32: Agarose gel electrophoresis: Restriction (Res) of the integrative
vector pBS1C with *Eco*RI and *Pst*I.**

The digested plasmid backbone results in a strong band at 6128 bp. The weak band at 1102 bp represents the RFP
insert being cut out of the vector.
The large band was purified by gel extraction and resulted in 35.2 ng/µl DNA. 1 kb Plus DNA Ladder (NEB)
served as marker (M).

After ligation, the plasmids were transformed into chemically competent *E. coli* DH10β cells
and transformants were selected by ampicillin resistance (100 μg/ml ampicillin) encoded on
the vector backbone. However, no colonies grew on selection plates, most likely due to very low transformation
efficiency.

As we already encountered these problems during phase 3 of our project, we repeated the
restriction of pBS1C with purification by PCR clean up instead of gel extraction (DNA concentration:
25.0 ng/µl, 29.2 ng/µl). Using this approach, we did not get rid of the original insert
containing RFP, but colonies with re-ligated plasmids appear pink on the plate due to the RFP
insert and can therefore be distinguished from correct transformants. Transformation plates
of spore display plasmids based on vector purification by PCR clean up are shown below (Fig. 33).

![](https://static.igem.wiki/teams/5117/images-wiki/results/16-e-coli-trafo-pbs1c-bhbgla-l1-l2-l3.webp)

**Figure 33: *E. coli* DH10β transformants on LB + ampicillin plates,
based on pBS1C purification by PCR clean up.**

Negative control (NC) containing no DNA showed no growth. Positive control (PC) containing pBS1C resulted in a
pink bacterial lawn.
Re-ligation control (RC) containing the digested pBS1C led to growth of approximately 400 pink and 20 white
colonies.
Transformation with plasmids pBS1C-P*cotYZ*-BhBglA-(L1/L2/L3)-*cotY*-B0014 resulted in ≈
300 colonies each, of which approximately half
appeared white and half appeared pink. However, white colonies are not necessarily correct as the re-ligation
control showed
few white colonies as well (barely visible in the photos).

White colonies transformed with the spore display plasmids were analyzed by Colony PCR and agarose
gel electrophoresis (Fig. 34). Colonies with a band of the correct size of the insert were chosen
for plasmid isolation and verification by sequencing. All plasmids of BsEglS, BhBglA and BhrPET
with three varying linkers were successfully generated.

For PpBglB, only the constructs with linkers L1 and L3 could be built. The PpBglB-L2 assembly
product resulted in white transformants with correct insert size after Colony PCR, but sequencing
revealed that none of the inserts had the correct sequence. After repeating ligation, *E. coli*
transformation and Colony PCR, no correct colonies could be found and, unfortunately, we could
not manage to generate this construct in time. Of the exoglucanases, only the pBS1C-P*cotYZ*-AtCelO-L2-*cotY*-B0014 plasmid could be
constructed due
to the cloning difficulties explained above.

![](https://static.igem.wiki/teams/5117/images-wiki/results/17-e-coli-colonypcr-pbs1c-bhbgla-l1-l2-l3.webp)

**Figure 34: Agarose gel electrophoresis: Insert amplification of pBS1C by
Colony PCR
of transformed *E. coli* DH10β cells, demonstrated with by BhBglA constructs.**

Oligonucleotides for amplification can be found on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page. Numbers 1-4 correspond to chosen
colonies.
Correct PCR products have a size of 2272 bp, 2326 bp and 2299 bp, depending on the linker (L1-L3). 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). Colonies 2 (BhBglA-L1), 1 (BhBglA-L2) and 1 (BhBglA-L3) were verified by
sequencing
and contained the correct insert sequence. Results for all parts are documented on the respective Parts
Registry
pages (links can be found on the [Contribution](https://2024.igem.wiki/tu-dresden/contribution)
page).

Ultimately, these plasmids had to be transformed into the wildtype strain W168 of our target host
*B. subtilis*. The protease-deficient strain WB800N was not required for the spore display approach
since proteins are not secreted in this case. Transformants were selected by chloramphenicol
resistance (5 μg/ml) encoded on the pBS1C vector backbone (Fig. 35).

![](https://static.igem.wiki/teams/5117/images-wiki/results/18-b-subtilis-trafo-pbs1c-bhbgla-l1-l2-l3.webp)

**Figure 35: *B. subtilis* W168 transformants on LB + chloramphenicol
plates.**

Negative control (NC) containing no DNA showed to no growth. Transformation is demonstrated with the
plasmids pBS1C-P*cotYZ*-BhBglA-(L1/L2/L3)-*cotY*-B0014, which were linearized by
restriction with *Bsa*I
prior to transformation and resulted in ≈ 400-500 white colonies each.

The successful genomic integration was verified via starch assay (Fig. 36). Four colonies per construct 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 were chosen for cryo-conservation.

![](https://static.igem.wiki/teams/5117/images-wiki/results/19-b-subtilis-starchassay-pbs1c-bhbgla-l1-l2-l3.webp)

**Figure 36: Starch assay of transformed *B. subtilis* W168 cells,
demonstrated with spore
display constructs of BhBglA with varying linkers (L1-L3).**

Numbers 1-4 correspond to chosen colonies. W168 served as control and displayed a bright halo. In contrast, no
halo
was visible for W168 strains with integrated spore display constructs (BhBglA-L1, BhBglA-L2, BhBglA-L3).
Results
for all parts are documented on the respective Parts Registry pages (links can be found on the [Contribution](https://2024.igem.wiki/tu-dresden/contribution) page).

In the end, we successfully generated 12 of 18 planned spore display plasmids (six
enzyme candidates: BsEglS, AtCelO, AtCelS, BhBglA, PpBglB, BhrPET, three linkers
each: L1, L2, L3) as well as their corresponding *E. coli* and *B. subtilis* strains
(Tab. 6), with the latter being used for subsequent activity tests.

Tab. 6: Spore display plasmids, *E. coli* DH10β and
*B. subtilis* W168 strains.

| Construct | DNA Concentration [ng/µl] | Plasmid No. | *E. coli* No. | *B. subtilis* No. |
| --- | --- | --- | --- | --- |
| **pBS1C-P*cotYZ*-BsEglS-L1-*cotY*-B0014** | 209.8 | P41 | Ec41 | Bs21 |
| **pBS1C-P*cotYZ*-BsEglS-L2-*cotY*-B0014** | 117.3 | P42 | Ec42 | Bs22 |
| **pBS1C-P*cotYZ*-BsEglS-L3-*cotY*-B0014** | 208.4 | P43 | Ec43 | Bs23 |
| **pBS1C-P*cotYZ*-BhBglA-L1-*cotY*-B0014** | 227.2 | P44 | Ec44 | Bs24 |
| **pBS1C-P*cotYZ*-BhBglA-L2-*cotY*-B0014** | 212.4 | P45 | Ec45 | Bs25 |
| **pBS1C-P*cotYZ*-BhBglA-L3-*cotY*-B0014** | 203.9 | P46 | Ec46 | Bs26 |
| **pBS1C-P*cotYZ*-PpBglB-L1-*cotY*-B0014** | 214.3 | P47 | Ec47 | Bs27 |
| **pBS1C-P*cotYZ*-PpBglB-L2-*cotY*-B0014** | - | - | - | - |
| **pBS1C-P*cotYZ*-PpBglB-L3-*cotY*-B0014** | 223.1 | P49 | Ec49 | Bs29 |
| **pBS1C-P*cotYZ*-BhrPET-L1-*cotY*-B0014** | 203.7 | P50 | Ec50 | Bs30 |
| **pBS1C-P*cotYZ*-BhrPET-L2-*cotY*-B0014** | 227.6 | P51 | Ec51 | Bs31 |
| **pBS1C-P*cotYZ*-BhrPET-L3-*cotY*-B0014** | 223.7 | P52 | Ec52 | Bs32 |
| **pBS1C-P*cotYZ*-AtCelO-L2-*cotY*-B00014** | 276.9 | P53 | Ec53 | Bs33 |

### Activity Assays of Enzymes Immobilized on *B.subtilis* Spores

Spores were prepared by culturing cells in LB medium with chloramphenicol until reaching the exponential
growth phase (OD600 of 0.4 – 0.6). After washing and resuspension in DSM, the culture was incubated
for
24 hours at 37 °C to induce sporulation. The cells were 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,
the pNPG assay, and the pNPAc assay, and to 2.8 for the DNS assay. For qualitative plate assays, an OD600 of
0.2 was used. Further details are available on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page.

**Endoglucanases**

To determine the CMCase activity of BsEglS displaying spores, we first performed a qualitative assay using
CMC-Agar plates following the protocol described on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page. 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. 37.

![](https://static.igem.wiki/teams/5117/images-wiki/results/6-endoglucanases.webp)

**Figure 37: Qualitative CMCase activity assay of BsEglS displaying spores on
CMC-agar plates.**

To assess CMCase activity, the optical density (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.

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 (L1, L2, L3) showed clear halos, demonstrating that enzyme activity was present.

To quantitatively compare the enzyme activities associated with the different linkers, we subsequently applied
the DNS assay, 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 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. 38. The resulted concentrations of reducing ends were 585.02 μg/ml, 616.81 μg/ml and
626.35 μg/ml
for BsEglS-L1, BsEglS-L2 and BsEglS-L3 respectively.

![](https://static.igem.wiki/teams/5117/images-wiki/results/7-endoglucanases.webp)

**Figure 38: DNS assay for determination of endoglucanase activity of BsEglS
with different linkers (L1, L2, L3).**

DNS assay was performed with BsEglS displaying spores (with final OD600 = 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 experiment was conducted with a single biological replicate (N = 1).

The assay was repeated with the reaction conducted for 30 minutes, as shown in Fig. 39. This time BsEglS
immobilized via
linker L2 showed the lowest activity which resulted in 101.67 μg/ml of produced reducing ends. BsEglS
immobilized via
linker L1 and L3 performed similarly, with BsEglS-L1 showing slightly higher activity. In samples with BsEglS-L1
293.33 μg/ml
of reducing ends were formed and with BsEglS-L3 275.00 μg/ml.

![](https://static.igem.wiki/teams/5117/images-wiki/results/8-endoglucanases.webp)

**Figure 39: DNS assay for determination of endoglucanase activity of BsEglS
with different linkers (L1, L2, L3).**

DNS assay was performed with BsEglS displaying spores (with final OD600 = 0.2 in reaction) in 50 mM phosphate
buffer pH 7 at 50 °C for 30 minutes
with two biological replicates. 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 experiment was conducted with two biological replicates (N = 2).

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.

To characterize the enzymes immobilized on spores, 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. 40). 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.

![](https://static.igem.wiki/teams/5117/images-wiki/results/9-endoglucanases.webp)

**Figure 40: Determination of temperature optimum of BsEglS displaying spores
with different linkers (L1, L2, L3)
by using DNS assay with OD600 of spore solution in reaction of 0.2.**

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 experiment was conducted with a single biological replicate (N = 1).

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. 41).
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.

![](https://static.igem.wiki/teams/5117/images-wiki/results/10-endoglucanases.webp)

**Figure 41: Determination of thermostability of endoglucanase displaying
spores (BsEglS-L1, BsEglS-L2, BsEglS-L3) by using DNS assay.**

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 experiment was conducted with a single 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.

**Exoglucanases**

To examine the functioning of AtCelO displaying spores (AtCelO-L2), a qualitative assay was performed using
PASC-Agar and PASA-Agar plates following the protocol
described on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page. Initially, the OD600
of spore solution was adjusted to 0.2, and 20 µL of it were pipetted onto the plates. The plates were incubated
at 50 °C for 24 hours (exemplary shown in Fig. 42). However, no activity could be detected afterwards. Different
OD600s, ranging from 0.5 to 4.0, were tested, as well
as the incubation at room temperature and 65 °C, but without success, indicating that *B. subtilis* has
struggles with the production of this enzyme.

![](https://static.igem.wiki/teams/5117/images-wiki/results/2-exoglucanases.webp)

**Figure 42: Qualitative PASC activity assay of AtCelO displaying spores
(AtCeO-L2) on PASC-agar plate to examine cellobiohydrolase activity.**

OD600 of the spore solution was adjusted to 0.2 and 20 µL of the suspension was applied to PASC-agar plates.

**β-Glucosidases**

To evaluate the performance of β-glucosidases displayed on spores, activity tests were conducted for
spore-displayed enzymes (BhBglA-L1, BhBglA-L2, BhBglA-L3, PpBglB-L1, PpBglB-L3) using pNPG and cellobiose as
substrates. Enzymatic activity was measured by monitoring absorbance at 405 nm for pNPG and 560 nm for
cellobiose
using the glucose assay. This approach allowed us to assess the catalytic efficiency of different linker
variants (L1, L2, L3) for the same enzyme, as well as compare the effectiveness of the two β-glucosidases,
BhBglA and PpBglB.

First, we prepared spores displaying BhBglA, as this enzyme showed promising results in previous assays
involving induced expression. We aimed to determine whether our glucose assay could effectively measure
the glucose concentration resulting from the degradation of 50 mM cellobiose. The assay was performed according
to the protocol described on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page,
using the Amplex™ Red Glucose/Glucose Oxidase Assay Kit.
After a 24-hour incubation period, the glucose assay was carried out, and the absorbance was measured at 560 nm.
The results are presented in Fig. 43.

As a control, 50 mM cellobiose, diluted in 1X reaction buffer, was used, and the substrate absorbance was
subtracted
from the measured values. All three linker variants (L1, L2, L3) exhibited comparable absorbance values of
approximately
0.2, corresponding to a glucose concentration of 13.8 µM, which, considering the dilution factor, results in
27.6 µM
in the reaction. These results suggest that there is no difference in glucose production between the
three
BhBglA linker variants, indicating similar catalytic efficiency for each. The glucose assay appears to be
effective,
although a relatively high background absorbance though unpurified cellobiose was observed (data not shown),
which
still allowed for distinguishing enzymatic activity from the control. In the future, we should further
investigate
cellobiose purification to reduce background signal of cellobiose.

![](https://static.igem.wiki/teams/5117/images-wiki/results/6-glucosidase.webp)

**Figure 43: Glucose concentration determination after degradation of 50 mM
cellobiose by spores displaying β-glucosidases (BhBglA-L1, BhBglA-L2, BhBglA-L3).**

The reaction with 50 mM cellobiose was incubated for 24 hours at 50 °C. Following incubation, the glucose
assay was performed using the Amplex™ Red Glucose/Glucose Oxidase Assay Kit,
and absorbance was measured at 560 nm. The spores of W168 strain were used as a negative control, while 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 the unpurified cellobiose. The experiment was conducted with a single biological replicate (N = 1).

We questioned whether a 24-hour incubation period was beneficial, given the low absorbance observed, 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
alongside spores displaying BhBglA-L2 to compare their activity. The
glucose assay was performed according to the protocol (see [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page), and the results are presented in
Fig. 44.

![](https://static.igem.wiki/teams/5117/images-wiki/results/7-glucosidase.webp)

**Figure 44: Glucose concentration determination following the degradation of
50 mM cellobiose by spores displaying β-glucosidases (BhBglA-L2, PpBglB-L3).**

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 of 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 value measured from cellobiose control was subtracted
from the enzyme activity measurements to account for the background signal of unpurified cellobiose. The experiment was conducted with a single biological replicate (N = 1).

W168 control showed no absorbance at all time points, indicating no glucose production and confirming the
absence of
enzymatic activity in the control spores. BhBglA-L2 displayed increasing absorbance values over time, with the
highest
absorbance at 20 minutes (approximately 0.18) and slightly lower at 30 minutes. This suggests effective
enzymatic activity,
though the slight decrease could indicate either substrate saturation or variability in the measurements due to
the lack of
triplicates. PpBglB-L3 showed no absorbance, comparable 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 of 20 minutes for
BhBglA-L2
is likely to reflect experimental fluctuations, as triplicates were not performed.

Based on previous results and the understanding that cellobiose might be difficult for these enzymes to degrade
efficiently,
we decided to use pNPG as a substrate to validate the findings from the glucose assay. The assay was performed
according to
the protocol outlined in the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) section. We
tested spores displaying BhBglA-L1, BhBglA-L2, BhBglA-L3, as well as
PpBglB-L1 and PpBglB-L3, in two biological replicates, to assess and compare their enzymatic activities using
pNPG as a more
accessible substrate. The results are shown in Fig. 45.

![](https://static.igem.wiki/teams/5117/images-wiki/results/8-glucosidase.webp)

**Figure 45: Evaluation of enzymatic activity of spore-displayed β-glucosidases
(BhBglA-L1, BhBglA-L2, BhBglA-L3, PpBglB-L1, PpBglB-L3) using pNPG as a substrate.**

The reaction was conducted at 50 °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 experiment was conducted with two biological replicates (N = 2). Measurements were performed in
duplicates, and mean values were calculated. The values were background corrected.

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 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 variants showed no 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, PpBglB was excluded from further testing due to the lack of observable
activity. Therefore, we focused on BhBglA
with three different linkers (L1, L2, L3). The optimal temperature for these was determined by evaluating enzyme
activity across a temperature range of
40 °C to 90 °C, using pNPG as the substrate to identify the temperature at which each construct displayed
maximal catalytic performance (as shown in Fig. 46).
The assay was conducted following the standard procedure, with the temperature varied between 40 °C and 90 °C
(see [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page).

![](https://static.igem.wiki/teams/5117/images-wiki/results/9-glucosidase.webp)

**Figure 46: Determination of the optimal temperature for spores displaying
β-glucosidase BhBglA with three different linkers (L1, L2, L3) using pNPG as a substrate.**

The reaction was conducted at different temperatures ranging from 40 °C to 90 °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, along with an 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 relative activity
is shown, with the highest value used for normalization, which was obtained from BhBglA-L2
at its maximum activity at 40 °C.

The W168 control shows no activity at any of the tested temperatures. At 40 °C, all three BhBglA linkers (L1,
L2, L3) exhibit relatively high activity, with L2 showing the highest activity,
followed by L1 and L3. The maximum activity for L1 and L2 is observed at 40 °C, while for L3, the maximum
activity is at 50 °C. There is a slight decrease in activity for L1 and L2 at 50 °C.
Generally, L3 shows higher activity than L1 and L2 at elevated temperatures. At 60 °C, the activity decreases
for all variants, but L3 retains the highest residual activity,
followed by L1 and L2. At higher temperatures (70 °C, 80 °C, and 90 °C), all immobilized enzymes show minimal
activity, indicating a sharp decline in enzyme performance, likely due to
denaturation. These results suggest that the optimal temperature for BhBglA activity varies between 40 °C and 50
°C depending on the linker, with activity diminishing above 60 °C. BhBglA-L3
appears to retain slightly higher stability at 60 °C compared to the other enzymes that are linked with L1 or
L2.

Additionally, the thermostability of the BhBglA variants was assessed by pre-incubating spore solutions at
temperatures ranging from 40 °C to 90 °C for 2 hours, followed by measuring
residual enzyme activity with pNPG (as shown in Fig. 47). This approach allowed us to evaluate the thermal
stability of the immobilized enzymes on spores fused with different linkers
by assessing how well each retained activity following heat exposure.

![](https://static.igem.wiki/teams/5117/images-wiki/results/10-glucosidase.webp)

**Figure 47: Determination of the thermostability of spores displaying
β-glucosidase BhBglA with three different linkers (L1, L2, L3) by using pNPG as a substrate.**

Before the reaction with pNPG, the spores were incubated at different temperatures ranging from
40 °C to 90 °C for 2 hours. Following this pre-incubation, the reaction with pNPG was conducted for
10 minutes at 50 °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 experiment
was conducted with a single biological replicate (N = 1). Measurements were performed in duplicates,
and mean values were calculated. The values were background corrected and normalized to the
corresponding values obtained without the pre-incubation of the spore solution at higher temperatures.

The relative activity of BhBglA (L1, L2, and L3) was analyzed after pre-incubation at various temperatures
ranging from 40 °C to 90 °C, followed by a reaction with pNPG for 10 minutes at 50 °C.
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.
After pre-incubation at 40 °C, all three tested immobilized enzymes exhibited some relative activity (25 % to 30
%), with BhBglA-L1 and BhBglA-L3 showing the highest values. In contrast,
no activity was observed for any of enzymes after pre-incubation at temperatures from 50 °C to 90 °C, suggesting
that the enzymes underwent heat-induced denaturation or lost their functional
capacity at these higher temperatures.

These findings indicate that BhBglA-L1 and BhBglA-L3 might be more thermostable than BhBglA-L2, as they retain
higher activity after exposure to 40 °C. However, above 40 °C, all tested
enzymes lose their activity, suggesting a temperature limit for functional stability due to heat-induced
denaturation. The W168 strain, used as a control, showed no activity
across all temperatures tested, confirming the specificity of the enzymatic function observed. These results
differ from previous literature, which reported an optimal temperature
of 45 °C for BhBglA activity, with the enzyme retaining 80% of its activity after incubation at 45 °C for 1 hour
(Naz *et al.*, 2010). The discrepancy might be attributed to
differences in enzyme immobilization and experimental setups, such as exposure time to heat.

The findings obtained also explain why we observed only minimal glucose production in the glucose assay
conducted over 24 hours. The rapid decline in enzyme activity at temperatures
above 40 °C likely limited the efficiency of cellobiose degradation, resulting in a low glucose yield.
Therefore, for future experiments, the degradation of cellobiose should be
carried out at 40 °C to ensure optimal enzyme performance. Additionally, the thermostability assessment should
be extended by incubating the spore solution at 40 °C for longer periods
prior to the reaction. This approach would provide a more comprehensive evaluation of enzyme stability and
suitability for industrial applications at this temperature. By understanding
the enzyme's long-term stability at 40 °C, it would be possible to determine whether it can sustain the desired
catalytic activity over prolonged industrial processes. It would also
be useful to investigate if the enzyme is active at lower temperatures and assess its stability under these
conditions. Evaluating the enzyme's activity and stability at temperatures
below 40 °C could help determine if the enzyme remains effective in milder conditions.

To strengthen the reliability of these findings, it is essential to perform these experiments in triplicates to
confirm the observed trends and ensure reproducibility of the results.

Further, we tested whether the spores interact with pNPAc, a substrate typically used to determine PETase
activity, to rule out any potential intraspecific enzymatic properties of
the spores themselves. This was done to ensure that any observed activity was due to the specific β-glucosidase
enzymes displayed on the spores and not inherent spore-associated
activity (as shown in Fig. 48).

![](https://static.igem.wiki/teams/5117/images-wiki/results/11-glucosidase.webp)

**Figure 48: Evaluation of spore interaction with substrates pNPG and pNPAc to determine specific enzyme activity.**

The reactions with pNPAc and pNPG were conducted at 40 °C and 50 °C respectively 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. The experiment was conducted with a
single biological replicate (N = 1). Measurements were performed in triplicates, and mean values were calculated.
The absorbance from the control without spores was subtracted from the measured values to account for
background signal. The amount of spores was adjusted to achieve an OD600 of 0.2 in the reaction mixture.

With pNPAc, the absorbance values are low and similar for all constructs, suggesting limited interaction, which
confirms that the activity
measured is specific to the β-glucosidase enzymes and not due to the inherent properties of the spores. Thus,
these results indicate
that spores displaying BhBglA exhibit specific enzymatic activity towards pNPG, while interaction with pNPAc is
minimal, ruling out
intraspecific enzymatic properties of the spores themselves. This supports the conclusion that the
observed activity
is due to the expressed β-glucosidase enzymes.

Finally, we evaluated the reusability of the spore-displayed enzymes for BhBglA fused with three different
linkers. The spores were
used in five reaction cycles, with a washing step between each cycle. The number of spores in the first cycle
was adjusted to achieve
an OD600 of 0.2 in the reaction mixture. The washing step involved removing the reaction products by ensuring the
spores settled at the
bottom of the reaction tube through centrifugation for 5 minutes at 13,000 rpm. The supernatant was discarded,
followed by the addition
of 1 ml of dH2O, another centrifugation step, and subsequent removal of the water. 100 µl of fresh
dH2O were added, and the spores were
stored until the next usage (20 minutes later). The reaction was conducted with pNPG as the substrate for 15
minutes instead of the
usual 10 minutes. After completing the final fifth cycle, the reaction mixture was measured, then incubated for
an additional 1 hour,
followed by another measurement (indicated as 5\* in Fig. 49).

The normalization was set to the first cycle (100 %) for each linker, and the subsequent cycles were normalized
relative to the first
cycle for each respective linker. The normalization for the control was set to BhBglA-L1 to ensure effective
comparison. The W168 control
showed no activity, confirming the absence of inherent enzymatic function. In the second cycle, a decrease in
activity was observed
for all linkers, with L1 retaining the highest activity (approx. 60 %), followed by L3 (approx. 50 %) and L2
(approx. 40 %). Activity
continued to decline across subsequent cycles, with L1 maintaining slightly higher activity in the third cycle
compared to L2 and L3.
By the fourth and fifth cycles, enzyme activity was minimal for all linkers, indicating a loss of catalytic
performance after repeated
use. After the additional 1-hour incubation in the fifth cycle (5\*), BhBglA-L1 showed an increase in activity
(up to 40 %), suggesting
some enzymatic potential during prolonged incubation. This may indicate slower catalytic degradation of pNPG due
to the partial loss of spores.

![](https://static.igem.wiki/teams/5117/images-wiki/results/12-glucosidase.webp)

**Figure 49: Evaluation of the reusability of spore-displayed BhBglA enzymes
with different linkers (L1, L2, L3) across five reaction cycles.**

Relative activity is shown, normalized to the first cycle (100 %) for each linker, with BhBglA-L1 used as the
reference for control
normalization. The spores underwent five reaction cycles, each followed by a washing step involving
centrifugation and resuspension
in dH2O. The reaction was conducted with pNPG as the substrate for 15 minutes. After the fifth
cycle, an additional measurement was
taken after 1-hour incubation (indicated as 5\*). The amount of spores in the first cycle was adjusted to
achieve an OD600 of 0.2 in the reaction mixture. The experiment was conducted with a single biological replicate (N = 1). Measurements were performed in duplicates.

These results indicate that while spores with immobilized BhBglA enzymes are reusable, there is a noticeable
decline in
activity with each reuse, likely due to enzyme inactivation or loss during the washing steps. BhBglA-L1
demonstrated relatively
better reusability, which might be coincidental, possibly due to reduced enzyme loss during washing.

The reusability of spore-displayed BhBglA with different linkers was assessed across five reaction cycles.
Although enzyme
activity decreased with each subsequent cycle, BhBglA-L1 showed relatively better performance,
with some recovery
observed after extended incubation in the fifth cycle. This suggests that while the spores are reusable, enzyme
inactivation or loss during washing impacts catalytic performance.

However, the OD600 of the spore solution was not measured during these experiments, which may have influenced the
results. In future experiments, it would be advisable to measure the OD600
and start with a higher value than the OD600 = 0.2 used in our experiments. This would allow for normalization to
OD600 and better management of spore loss during the washing steps,
leading to more reliable and comparable results across different reaction cycles.

**PETase**

The PETase activity of spores displaying BhrPET with three different linkers (L1, L2, L3) was evaluated using an
activity assay based on the method described by
Xi *et al.* (2021). 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 [Experiments](https://2024.igem.wiki/tu-dresden/experiments) 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. 50.
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.

![](https://static.igem.wiki/teams/5117/images-wiki/results/2-petase.webp)

**Figure 50: Activity assay of BhrPET-displaying spores with different
linkers (L1, L2, L3) after 10 minutes of incubation at 40 °C with pNPAc as the substrate.**

Absorbance was measured at 405 nm using 100 µl of the reaction supernatant. The amount of spores was
adjusted to achieve an OD600 of 0.2 in the reaction mixture. The experiment was conducted with a single
biological replicate (N = 1). Measurements were performed in triplicates, 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 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. 51 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).

![](https://static.igem.wiki/teams/5117/images-wiki/results/3-petase.webp)

**Figure 51: Activity assay of BhrPET-displaying spores with different linkers
(L1, L3) after 10 minutes of incubation at 40 °C with pNPAc as the substrate.**

Absorbance was measured at 405 nm using 100 µl of the reaction supernatant. The amount of spores was adjusted
to achieve an OD600 of 0.2 in the reaction mixture.
Measurements were performed in technical triplicates, and the mean of both biological replicates (N = 2) is shown. The
control included a reaction with W168 spores,
which did not display any enzyme, as well as a reaction without spores, which was subtracted as the blank. The
data represent absorbance values with control
subtracted, indicating the relative enzyme activity of the different constructs.

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.

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. 52.

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.

![](https://static.igem.wiki/teams/5117/images-wiki/results/4-petase.webp)

**Figure 52: Relative activity of BhrPET-L1 and W168 spores at varying
temperatures (40 °C to 90 °C) for determining the optimal temperature for enzyme activity.**

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. 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 experiment was conducted with a single biological replicate (N = 1). Measurements were performed in duplicates, and mean values were calculated.

Spore solutions were incubated at various temperatures ranging from 40 °C to 90 °C for 2 hours to evaluate the
thermostability of the enzyme displayed on the spores. After heat treatment, the samples were allowed to cool to
room temperature. Subsequently, the residual enzyme activity of BhrPET-L1 was determined using an assay previously
described (for 10 minutes at 40 °C). 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 results of this assessment
are presented in Fig. 53.

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 drastically diminish at temperatures above 60 °C.

![](https://static.igem.wiki/teams/5117/images-wiki/results/5-petase.webp)

**Figure 53: 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.**

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 conducted with a single biological replicate (N = 1). Measurements were performed in duplicates, and mean values were calculated.

Noteworthy, the obtained results contradict earlier findings from the work of Xi *et al.* (2021), 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.* (2021) 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 drastic 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.* (2021) 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.

**MILESTONE 3:**

**Generation of *B. subtilis* spores with active immobilized enzymes**

**Endoglucanase: BsEglS**

**β-Glucosidase: BhBglA**

**PETase: BhrPET**

---

## Conclusion

### Project Summary

During this project, we successfully implemented our two strategies of induced expression
and spore surface display. In the induced expression reference approach, all enzyme
candidates were successfully cloned into both replicative and integrative expression plasmids,
allowing for the overexpression of target genes.

Despite some difficulties during the initial activity assays, we ultimately developed functional
assays for endoglucanases, exoglucanases, β-glucosidases, and PETases. Enzyme activity was detected
for endoglucanases (BsEglS, BpEglA, AtCelA), PETase (BhrPET) and β-glucosidases (BhBglA). Even though
we did not find any activity with β-glucosidase (PpBglB), we proceeded with spore surface display
for this enzyme based on supporting literature. No activity was observed for the exoglucanases
(AtCelO, AtCelS). The lack of activity for exoglucanases could be due to several factors, such as
improper folding, insufficient expression levels, or instability of the enzyme under assay conditions.
Additionally, the activity observed for enzymes expressed using replicative vectors was higher than
that observed for those using integrative vectors; however, not many tests were conducted in this
area due to time constraints.

Functional enzymes were chosen for our final spore surface display strategy. Selected enzymes were fused to an
anchor protein within the Bacillus spore crust, whereby flexible (L1, L2) and rigid (L3) linkers were tested.
Spore display plasmids of the endoglucanase (BsEglS), β-glucosidases (BhBglA and PpBglA, excluding PpBglB-L2)
and PETase (BhrPET) were successfully generated. We were unable to construct exoglucanases in time (except
AtCelO-L2)
due to difficulties that occurred during cloning.

Ultimately, *B. subtilis* spores with immobilized enzymes were analyzed. The endoglucanase BsEglS, the
β-glucosidase
BhBglA, and the PETase BhrPET all displayed activity. Additionally, a reusability assay for the β-glucosidase
BhBglA
was conducted, providing promising results regarding the sustainability of our project.

The characterization of enzymes immobilized on spores revealed that the optimal temperature for activity varied
among the
studied enzymes. For BsEglS, the highest activity was observed at 60 °C, consistent with earlier findings that
suggested an
optimal temperature of 65 °C, indicating that immobilization did not affect the optimal temperature. Similarly,
BhrPET and BhBglA demonstrated optimal activity at 40 °C to 50 °C.

The thermostability analysis showed that all enzymes experienced a decline in activity at temperatures above 60
°C, likely
due to heat-induced denaturation. BhrPET-L1 retained activity up to 60 °C, while BhBglA variants lost activity
after
pre-incubation above 40 °C. The control strain W168 exhibited no activity, confirming the
specificity of the
enzymatic activity observed. These findings indicate that while the immobilization of endoglucanases and other
enzymes on
spores provided functional enzymes, their thermostability did not improve beyond their natural temperature
limits.

All laboratory work is visualized on the [Timeline](https://2024.igem.wiki/tu-dresden/timeline) page,
providing an overview of all lab activities carried out during this summer.

### Project Achievements

Although we encountered difficulties in the lab that led to some unsuccessful results, we also experienced many
successes, all of which are summarized below.

Tab. 7: Project achievments.

| Successful Results | Unsuccessful Results |
| --- | --- |
| **Induced Expression**   * Generation of replicative and integrative expression  plasmids as well as *E. coli* and *B.   subtilis* strains for  all 10 enzyme candidates * Endoglucanases (BsEglS, BpEglA, AtCelA),  β-glucosidases (BhBglA) and PETase (BhrPET)  are   functional in *B. subtilis*. | **Induced Expression**   * Generation of *B. subtilis* WB800N deletion strains (Δ*eglS*, Δ*bglH*) to remove   background activity failed * No detection of enzyme activity for exoglucanases  (AtCelO, AtCelS), for endoglucanase (AtCelG)  and   β-glucosidases (PpBglB, AtBglA) |
| **Spore Surface Display**   * Generation of spore display plasmids as well as  *E. coli* and *B. subtilis* strains for   chosen enzymes (BsEglS-L1/L2/L3, AtCelO-L2, BhBglA-L1/L2/L3, PpBglB-L1/L3,  BhrPET-L1/L2/L3) * Endoglucanase BsEglS, β-glucosidase BhBglA and PETase BhrPET are active on *B. subtilis*   spores * *B. subtilis* spores with immobilized enzymes can be reused several times | **Spore Surface Display**   * No construction of spore display plasmids for  exoglucanases (AtCelO and AtCelS, except AtCelO-L2) * No activity of β-glucosidase PpBglB-L1/L3 was detected * No increase of thermostability due to immobilization on spores * We did not manage to degrade actual textile fibers or PET |

---

## Outlook

### Considerations for Replicating Experiments

In both of our strategies (induced expression and spore surface display), we tested both clones of our generated
*B. subtilis* strains only once. Typically, the experiments were
performed
in a single replicate, with few exceptions (such as spore-displaying endoglucanases and β-glucosidases).

All constructed strains, however, were always stored in biological duplicates (clones 1 and 2), which provides
the
opportunity to test both clones in future experiments. Additionally, three independent biological experiments
should be conducted for each strain to obtain three replicates. Furthermore, all measurements should have been
performed in triplicates for better accuracy.

All information and protocols required for replicating experiments are documented on the [Experiments](https://2024.igem.wiki/tu-dresden/experiments) page.
If future iGEM teams would like to reproduce our experiments and need additional details, please do not hesitate
to contact us: **igem@tu-dresden.de** or our principal investigator Prof. Thorsten Mascher:
**thorsten.mascher@tu-dresden.de**.

### Characterization of Enzymes

During this project, various methods and strategies were employed to characterize and improve the
expression and activity of several enzymes. However, there are several areas that could be refined and
improved in future studies to ensure more reliable and informative results.

The optimization of protein yield obtained after expression in *B. subtilis* is still possible.
For that, e.g., the influence of different promoters on protein production should be explored.
Furthermore, in the future, the expression of proteins should be repeated under different conditions,
such as varying temperature and incubation times, to determine optimal expression time and settings.

The use of SDS-PAGE proved useful for determining the molecular weight of expressed proteins,
as described in the results section. However, the SDS-PAGE experiments should have been repeated,
as some gels were of poor quality. Furthermore, the purification of lysates and supernatants would
likely lead to better and more distinct results in SDS-PAGE, enhancing the accuracy of our molecular weight
assessments.

The optimal pH and pH stability of the spore-displaying enzymes should be determined to further
characterize their performance. Testing these parameters will help understand how the enzymes behave
under different environmental conditions, which is essential for potential industrial applications. Further
optimization of assay conditions could be obtained by testing different OD600 values as well as substrate
concentrations. This could also help to determine enzyme substrate specificity (e.g., as Km value).
Additionally, determining the actual amount of enzymes displayed on the spore surface is crucial for better
activity assessment and comparison with other studies.

The reusability assay of immobilized β-glucosidase BhBglA showed promising results, indicating the potential
for sustainable application. However, a better protocol for purification and yield of spores is required to
improve reproducibility. Starting with a higher OD600 or adjusting substrate concentration may help optimize
these experiments and yield better enzyme activity.

The glucose assay was used throughout this study; however, the assay has not been fully implemented due
to high background signal from cellobiose. Attempts were made to purify cellobiose, but these were unsuccessful
(see [Engineering](https://2024.igem.wiki/tu-dresden/engineering) page). Tests were also conducted to
optimize the assay using lysate, but without purification
of the lysate, the results were unreliable. The purification of cellobiose using glucose oxidase could be
considered,
though issues with interference in the glucose assay must be addressed. Other coupled assays may provide more
accurate insights into enzyme activity.

Overall, the experiments have shown promising results, especially for the enzymes BsEglS, BhBglA, and BhrPET.
However, further optimization and detailed characterization are required to better understand and improve the
efficiency of the enzymatic activity and spore immobilization processes, as well as to upscale this project for
industrial applications.

### Future Plans

Regarding cloning, all remaining spore display constructs must be generated, including PpBglB-L2,
AtCelO-L1, AtCelO-L3 as well as all three AtCelS constructs. Moreover, we only constructed N-terminal
fusions of the enzymes to the anchor protein. C-terminal fusions can also be tested in future experiments.
All following transformation experiments and activity assays need to be performed.

The activity assay for exoglucanases should also be further developed, as we continue to encounter
difficulties in detecting any activity from the enzyme candidates. If the tested exoglucanase candidates
AtCelO and AtCelS prove to be non-functional in *B. subtilis*, alternative enzymes will need to be
identified through literature research.

Finally, when one active enzyme from each category (endoglucanase, exoglucanase, β-glucosidase, PETase) will be
identified,
these enzymes can be immobilized on one spore by fusing them to different anchor proteins of the spore coat.
Many spore coat
proteins, e.g. CotY, CotB, CotC and OxdD, have already been used for protein immobilization (Zhang *et al.*,
2019; Lin *et al.*, 2020),
making the *B. subtilis* spore a three-dimensional immobilization platform. However, it is still unknown
whether this approach
will retain functional enzymes due to possible interaction between the immobilized proteins resulting in mutual
inhibition
and loss of activity. Nevertheless, the enzymes BhBglA, BsEglS as well as BhrPET are promising candidates for
this strategy.

Following the successful application of this approach, the degradation of actual textile fibers needs to be
tested. For that purpose,
extensive experiments must be performed regarding the pretreatment of textile waste and subsequent degradation
ability of our spores.

To successfully upscale our project from the lab to an industrial level, we would need to ensure safety and
compliance with
regulations. While modifying the spores to lack the ability to germinate is not strictly necessary for
industrial use, it
would be required if we wanted to avoid an S1 (biosafety level 1) classification. To proceed with this approach,
we would
first need to consult with the ZKBS (Central Committee on Biological Safety) to determine their specific
requirements.
Combining approaches, we could consider using the non-germinating strains engineered by the
[LMU Munich iGEM 2012](https://2012.igem.org/Team%3ALMU-Munich) team and transform them with our spore
display constructs in future experiments.

If the creation of *B. subtilis* spores unable to germinate and carrying active textile degrading enzymes
is successful at lab scale,
our project could finally be transferred to industrial scale by testing these spores in large bioreactors (see
[Entrepreneurship](https://2024.igem.wiki/tu-dresden/entrepreneurship) page).

---

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