Part:BBa_K5117024

BsEglS-L2

This part contains the eglS gene of Bacillus subtilis excluding its native signal peptide for secretion, encoding an endoglucanase (EC 3.2.1.4).

Downstream of the coding sequence, a long flexible linker (L2) has been added encoding the amino acids (GGGGS)4.

BsEglS-L2 only served for design purposes of the TU Dresden iGEM 2024 Team and was required for the construction of composite parts (see Contribution).

Target organism: Bacillus subtilis

Main purpose of use: Gene expression and production of fusion proteins (especially for spore surface display)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 526
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design

For compatibility with the BioBrick RFC[10] standard, the restriction sites EcoRI, XbaI, SpeI, PstI and NotI were removed from the coding sequence (CDS). To make the part compatible with the Type IIS standard, BsaI and SapI sites were removed as well. This was achieved by codon exchange using the codon usage table of Bacillus subtilis (Codon Usage Database Kazusa).

BsEglS-L2 is designed to be fused to the N-terminus of another protein. Therefore, the coding sequence does not contain a stop codon. Moreover, different linkers between the fused target enzyme and following protein can be analyzed, as these proteins may affect the folding and stability of each other and, eventually, lead to misfolding and reduced activity. Whereas flexible linkers promote the movement of joined proteins and are usually composed of small amino acids (e.g. Gly, Ser, Thr), rigid linkers are usually applied to maintain a fixed distance between the domains (Chen et al. 2013).

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

The part BsEglS-L2, documented in this page, contains the long flexible linker (GGGGS)4.

Enzyme characterization according to literature

In the study by Kari Aa, Ragnar Flengsrud, Viggo Lindahl, and Arne Tronsmo (1994), titled "Characterization of production and enzyme properties of an endo-β-1,4-glucanase from Bacillus subtilis CK-2 isolated from compost soil", the authors investigated the properties of an endo-β-1,4-glucanase enzyme produced by Bacillus subtilis CK-2 (Aa et al. 1994).

Bacillus subtilis CK-2, isolated from composted garden organic waste, produces endo-β-1,4-glucanase that exhibits high hydrolytic activity against carboxymethylcellulose (CMC). The production of this enzyme is associated with the bacterium's sporulation process and is regulated by the concentration of readily metabolizable carbohydrates in the growth medium. The induction of enzyme production does not require the presence of CMC or other cellulose-containing materials (Aa et al. 1994).

The enzyme activity was assessed by incubating diluted enzyme solutions with 1% CMC in various buffers covering a pH range from 3.2 to 9.6 for 30 minutes. The purified endo-β-1,4-glucanase demonstrated optimal activity over a broad pH spectrum, achieving peak performance at pH 5.6 in citrate/phosphate buffer and at pH 5.8 in phosphate buffer (Aa et al. 1994).

Temperature profiling revealed that the enzyme reaches maximum activity at 65 °C. The enzyme's activity was evaluated at temperatures ranging from 10 °C to 80 °C for a 30-minute incubation period, followed by a standard activity assay at 50 °C to determine residual activity. Thermal stability tests indicated that the enzyme retains its activity up to 55 °C but loses functionality when exposed to temperatures above this threshold for 30 minutes (Aa et al. 1994).

Molecular weight analysis through SDS-PAGE showed that the enzyme is a monomer with an approximate size of 35.5 kDa. In contrast, Sephadex G-75 chromatography suggested the presence of an active dimeric form with a molecular weight around 70 kDa, indicating that the active enzyme may function as a dimer (Aa et al. 1994).

More information related to this part can be found in the following publications and databases:

• MacKay R. M., Lo A., Willick G., Zuker M., Baird S., Dove M., Moranelli F., Seligy, V. (1986): Structure of a Bacillus subtilis endo-β-l, 4-glucanase gene. Nucleic acids research 14(22), 9159-9170. https://doi.org/10.1093/nar/14.22.9159
• Lindahl V., Aa K., Tronsmo A. (1994): Nucleotide sequence of an endo-β-1, 4-glucanase gene from Bacillus subtilis CK-2. Antonie van Leeuwenhoek 66, 327-332. https://doi.org/10.1007/BF00882768
• Gene sequence: https://www.ncbi.nlm.nih.gov/gene/938607
• UniProtKB: https://www.uniprot.org/uniprotkb/P10475/entry

References

Aa K., Flengsrud R., Lindahl V., Tronsmo A. (1994): Characterization of production and enzyme properties of an endo-β-1, 4-glucanase from Bacillus subtilis CK-2 isolated from compost soil. Antonie Van Leeuwenhoek 66, 319-326. https://doi.org/10.1007/BF00882767

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