Part:BBa_K5117035

PcotYZ-BsRBS-BsEglS-L1-CotY-B0014

This part serves as transcriptional unit composed of:

- promoter PcotYZ of Bacillus subtilis (BBa_K5117021),

- ribosome binding site of Bacillus subtilis (BBa_K5117000),

- eglS gene of Bacillus subtilis without signal peptide encoding an endoglucanase (EC 3.2.1.4),

addition of a short flexible linker (L1) downstream of the coding sequence encoding the amino acids GA (BBa_K5117023),

- cotY gene of Bacillus subtilis (BBa_K5117022),

- bidirectional terminator B0014 (BBa_B0014).

Biosafety level: S1

Target organism: Bacillus subtilis

Main purpose of use: Immobilization of BsEglS on the spore crust of B. subtilis (spore surface display)

Application: degradation of cellulose

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 NgoMIV site found at 1642
  Illegal AgeI site found at 726
  
• 1000
  COMPATIBLE WITH RFC[1000]

Enzyme characterization according to literature

The characterization of the enzyme included in this composite part can be found on the basic part page (BBa_K5117023) of the enzyme.

Construct 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).

To express target genes only under sporulation, the sporulation-dependent promoter P</sub>cotYZ</sub> of B. subtilis was chosen. In previous studies, this promoter has so far provided the highest activity for spore surface display (Bartels et al. 2018, unpublished data of Elif Öztel). The promoter was followed by the ribosome binding site (RBS) for the host B. subtilis with a 7 bp spacer.

To anchor the target enzyme on the spore surface, it was fused to the N-terminus of the anchor protein CotY. This anchor is located in the crust, the outermost spore layer, and has been shown to be well suited for protein immobilization (McKenney et al. 2013, Bartels et al. 2018, Lin et al. 2020).

Moreover, different linkers between the fused target enzyme and anchor protein were 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 composite part documented in this page contains the short flexible linker GA.

Following the CDS of cotY, a spacer consisting of 10 bp of the natural genome sequence downstream from the cotYZ operon was inserted. This creates space before the terminator and ensures that the ribosome is able to read the full length of the CDS. The construct ends with the terminator B0014, a bidirectional terminator consisting of B0012 and B0011.

The entire construct was flanked with the BioBrick prefix and suffix, allowing for cloning via the BioBrick assembly standard and restriction-ligation-cloning. The vector (pBS1C) from the Bacillus BioBrickBox was used as an integrative plasmid backbone enabling genomic integration into the amyE locus of B. subtilis (Radeck et al. 2013).

Construction of spore display plasmids

First, all biological parts including the enzyme candidate (Fig. 1) as well as the promoter P_cotYZ, the terminator B0014 and the anchor gene cotY (Fig. 2) were amplified by PCR and purified using the HiYield® PCR Clean-up/Gel Extraction Kit (SLG, Germany). The RBS was added by oligonucleotides. The plasmid pSB1C3-BhBglA generated in the subcloning phase served as template for PCR of the enzyme candidate (see (BBa_K5117011)). The linker was added by oligonucleotides. The promoter and anchor gene were amplified from genomic DNA of B. subtilis W168 and the terminator from a plasmid provided by the laboratory collection of Prof. Thorsten Mascher (General Microbiology, TU Dresden).