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PROJECT

Description Contribution Project Modeling Engineering Success Implementation

HUMAN PRACTICES

Human Practices Safety

TEAM

Team members Attributions Collaborations Science Communications

PARTS NOTEBOOK

PROJECT

Description Contribution Project Modeling Engineering Success Implementation

HUMAN PRACTICES

Human Practices Safety

TEAM

Team members Attributions Collaborations Science Communications

PARTS NOTEBOOK

OUR PROJECT

BBa_K3493000

Our team will be using a dextranase from a psychrophile organism (Gelidibacter algens) to degrade dextran in ropy maple syrup. Since we will be expressing this protein in Escherichia coli, we optimized its codon usage accordingly. This part is available in the iGEM registry under code BBa_K3493000. Following reports that the Streptococcus mutans homologous dextranase has a higher activity when its N terminal (residues 1-99) and C-terminal (residues 733-850) are deleted (Kim et al., 2011), we removed them from the CDS submitted to the registry. Accordingly, our final CDS contains the region of the G. algens dextranase sequence that aligns to residues 104 - 734 of the PDB structure of the S. mutans dextranase (Suzuki et al., 2012). This enzyme will be inserted in a pET-28a vector for its expression in E. coli (Figure 1).

Figure 1. Plasmid map for pET-28a after inserting the CDS of the G.algens dextranase (part BBa_K3493000). Figure generated with Snapgene.

BBa_K3493001

We identified a second candidate in the Aureobasidium subglaciale dextranase. However, A. subglaciale is an eukaryotic organism, which could pose problems for its proper expression in E. coli. We decided to save this candidate as a backup plan for further testing in case we had problems with our main candidate enzyme. Accordingly, we optimized its codon usage for E. coli and submitted the sequence to the iGEM registry under code BBa_K3493001.

References

• Kim, Y.-M., Shimizu, R., Nakai, H., Mori, H., Okuyama, M., Kang, M.-S., Fujimoto, Z., Funane, K., Kim, D., & Kimura, A. (2011). Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity. Applied Microbiology and Biotechnology, 91(2), 329–339.
• Suzuki, N., Kim, Y.-M., Fujimoto, Z., Momma, M., Okuyama, M., Mori, H., Funane, K., & Kimura, A. (2012). Structural elucidation of dextran degradation mechanism by streptococcus mutans dextranase belonging to glycoside hydrolase family 66. The Journal of Biological Chemistry, 287(24), 19916–19926.

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