Difference between revisions of "Part:BBa K5117026"

Revision as of 19:03, 30 September 2024

BhBglA-L1

This part contains the bglA gene of Bacillus halodurans, encoding a β-glucosidase (EC 3.2.1.21).

Downstream of the coding sequence, a short flexible linker (L1) has been added encoding the amino acids GA.

BhBglA-L1 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

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).

BhBglA-L1 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 BhBglA-L1, documented in this page, contains the short flexible linker GA.

Enzyme characterization according to literature

In the study by Nas et al. (2010) titled "Enhanced production and characterization of a beta-glucosidase from Bacillus halodurans expressed in Escherichia coli", the production and characterization of the enzyme β-glucosidase A (BglA) enzyme from Bacillus halodurans was explored through heterologous expression in E. coli (Naz et al. 2010).

The recombinant plasmid pET-BglA was transformed into E. coli BL21(DE3) CodonPlus cells. The induced protein was detected by SDS-PAGE, where a prominent band corresponding to the expected molecular weight of ~51 kDa was observed (Naz et al. 2010).

Cells were harvested, sonicated, and the soluble fraction was precipitated using ammonium sulfate. Purification was performed using anion exchange chromatography. The fractions were analyzed for BglA activity, yielding a purified enzyme for further characterization. BglA displayed the highest activity at pH 8.0, using glycine-NaOH buffer. BglA was stable at pH 7.5 – 8.0 at 45 °C for one hour, but stability decreased significantly at higher pH levels (Naz et al. 2010).

The enzyme retained more than 90% residual activity when incubated at pH 7.5-8.0 at 45 °C for one hour, but activity sharply decreased at pH > 8.0, dropping to 20% residual activity at pH of 9.5. The optimal temperature for BglA activity was found to be 45 °C when assayed using p-nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate. The enzyme retained 80 % of its activity when incubated at temperatures up to 45 °C for 1 hour, but activity decreased significantly at higher temperatures. BglA retained 80 % activity when incubated at 45 °C for one hour, but a decline in activity was observed with longer incubation times or at higher temperatures (Naz et al. 2010).

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

• Takami, H., Nakasone, K., Takaki, Y., Maeno, G., Sasaki, R., Masui N., Fuji F., Hirama C., Nakamura Y., Ogasawara N., Kuhara S., Horikoshi, K. (2000): Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic acids research 28(21), 4317-4331. https://doi.org/10.1093/nar/28.21.4317
• Gene sequence: https://www.ncbi.nlm.nih.gov/nuccore/BA000004
• Protein sequence: https://www.ncbi.nlm.nih.gov/protein/BAB05642
• UniProtKB: https://www.uniprot.org/uniprotkb/Q9KBK3/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

Naz S., Ikram N., Rajoka M. I., Sadaf S., Akhtar M. W. (2010): Enhanced production and characterization of a β-glucosidase from Bacillus halodurans expressed in Escherichia coli. Biochemistry (Moscow) 75, 513-518. https://doi.org/10.1134/s0006297910040164