Difference between revisions of "Part:BBa K5117030"

Latest revision as of 00:09, 2 October 2024

PpBglB-L2

This part contains the bglB gene of Paenibacillus polymyxa (synonym Bacillus polymyxa), encoding a β-glucosidase (EC 3.2.1.21).

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

PpBglB-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 PpBglB to the N-terminus of another protein for working in B. subtilis

Potential application: Spore surface display

Sequence and Features

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).

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

Enzyme characterization according to literature

In the study titled "Sequences and homology analysis of two genes encoding β-glucosidases from Bacillus polymyxa" (González-Candelas et al. 1990), the nucleotide sequences of the bglA and bglB genes were determined. These genes encode two β-glucosidase enzymes, each with coding regions of 1344 bp, corresponding to polypeptides with molecular weights of 51.6 kDa and 51.5 kDa, respectively (González-Candelas et al. 1990).

In a more recent study titled "One-step purification and characterization of β-glucosidase enzyme from Paenibacillus polymyxa" (Tsabitah et al. 2024), the researchers focused on the purification and characterization of recombinant β-glucosidase from Paenibacillus polymyxa. The enzyme was expressed heterologously in E. coli BL21 (DE3) and purified using a one-step 6x-His tag purification method (Tsabitah et al. 2024).

The enzyme was successfully purified in a single step. SDS-PAGE analysis revealed a single band corresponding to a molecular weight of 52 kDa, confirming the enzyme’s purity. The specific activity of the purified enzyme was measured using the p-nitrophenol β-D-glucopyranoside (pNPG) assay, yielding a specific activity of 4.1 U/mg. The enzyme displayed optimal activity at pH 6.0. The enzyme was most active at 55°C. The enzyme activity was affected by metal ions, with Zn²⁺ showing the most significant effect on its activity (Tsabitah et al. 2024).

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

• Isorna, Pablo et al., beta-glucosidase B from Paenibacillus polymyxa (2006) https://doi.org/10.2210/pdb2O9P/pdb
• Isorna P., Polaina J., Latorre-García L., Cañada F. J., González B., Sanz-Aparicio, J. (2007): Crystal structures of Paenibacillus polymyxa β-glucosidase B complexes reveal the molecular basis of substrate specificity and give new insights into the catalytic machinery of family I glycosidases. Journal of molecular biology 371(5), 1204-1218. https://doi.org/10.1016/j.jmb.2007.05.082
• Gene sequence: https://www.ncbi.nlm.nih.gov/nuccore/M60211
• Protein sequence: https://www.ncbi.nlm.nih.gov/protein/AAA22264
• UniProtKB: https://www.uniprot.org/uniprotkb/P22505/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

González-Candelas L., Ramón D., Polaina J. (1990): Sequences and homology analysis of two genes encoding β-glucosidases from Bacillus polymyxa. Gene 95(1), 31-38. https://doi.org/10.1016/0378-1119(90)90410-s

Tsabitah K., Saksono B., Khayyira A. S., Zulfa A., Ubaidillah M., Ermawar R. A. (2024): One-step purification and characterization of β-glucosidase enzyme from Paenibacillus polymyxa. AIP Conference Proceedings 2973 (1). https://doi.org/10.1063/5.0184761