Part:BBa_K5117047

AtCelO-L2

This part contains the celO gene of Acetivibrio thermocellus (synonym Clostridium thermocellum) excluding its native signal peptide for secretion, encoding an exoglucanase (EC 3.2.1.176).

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

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

Biosafety level: S1

Target organism: Bacillus subtilis

Main purpose of use: Fusion of AtCelO to the N-terminus of another protein for working in B. subtilis

Potential application: Spore surface display

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 984
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 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).

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

Enzyme characterization according to literature

In the study of Zverlov et al. titled “A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose”, the structure of the celO gene from Clostridium thermocellum F7 is reported and the corresponding protein was shown to possess cellobiohydrolase activity (Zverlov et al. 2002).

Two truncated proteins were constructed and examined: rCelO, with the leader peptide and the dockerin module deleted (587 aa, 67.3 kDa), and rCelO-Cat, representing only the catalytic domain of CelO (415 aa, 47.9 kDa). The resulting enzymes were recombinantly produced in Escherichia coli and purified via 6x-His tag purification method. Using barley β-glucan as substrate, the optimal temperature and pH were determined to be 65 °C and 6.6, respectively (Zverlov et al. 2002).

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

• Ahmad S., Sajjad M., Altayb H. N., Sarim Imam S., Alshehri S., Ghoneim M. M., Shahid S., Mirza M. U., Nadeem M. S., Kazmi I., Waheed Akhtar, M. (2023): Engineering processive cellulase of Clostridium thermocellum to divulge the role of the carbohydrate‐binding module. Biotechnology and Applied Biochemistry 70(1), 290-305. https://doi.org/10.1002/bab.2352
• Gene sequence: https://www.ncbi.nlm.nih.gov/nuccore/AJ275975
• Protein sequence: https://www.ncbi.nlm.nih.gov/protein/CAB76938
• UniProtKB: https://www.uniprot.org/uniprotkb/Q9L3J2/entry

References

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

Zverlov V. V., Velikodvorskaya G. A., Schwarz W. H. (2002): A newly described cellulosomal cellobiohydrolase, CelO, from Clostridium thermocellum: investigation of the exo-mode of hydrolysis, and binding capacity to crystalline cellulose. Microbiology 148(1), 247-255. https://doi.org/10.1099/00221287-148-1-247