Coding

Part:BBa_K5117024

Designed by: Jenny Sauermann, Lilli Kratzer, Katrin Lehmann   Group: iGEM24_TU-Dresden   (2024-09-28)
Revision as of 08:50, 30 September 2024 by Jesa98 (Talk | contribs)


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

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


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