Difference between revisions of "Part:BBa K5117001"

Latest revision as of 23:35, 1 October 2024

BsEglS

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

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

Biosafety level: S1

Target organism: Bacillus subtilis

Main purpose of use: Expression in the host B. subtilis

Potential application: Degradation of cellulose

Design

For compatibility with the BioBrick RFC[10] standard, the restriction sites EcoRI, XbaI, SpeI, PstI and NotI were removed from the 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).

Sequence and Features

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