During our project, we created a diverse library of parts, most of which code for biosurfactant proteins. We chose to focus mainly on the expression of hydrophobins from Trichoderma reesei and Grifola frondosa, which have demonstrated emulsion-formation properties1, a key step in the uptake of waste cooking oil by Yarrowia lipolytica2. The parts were synthesized as gene blocks by IDT and were assembled into pET28 or pET29 backbones to create expression plasmids in E. coli or into a pBEVY backbone for S. cerevisiae cloning. We also obtained parts for cloning in Yarrowia and generated in silico constructs based on an NDV-URA3 plasmid backbone.
Our personal favorite, BBa_K4661015, takes the expression of hydrohobin sequences to the next level. This feat is accomplished through engineered mutations that improve the native protein’s solubility and secretory capability in E. coli, as demonstrated by the literature3. Moreover, in this part we created a fusion between the mutated HFBI sequence and sfGFP, for better part characterization and expression validation. For more information, please visit the iGEM Part Registry.
Below you can find a list of all the other parts we created.
| Name | Type | Description |
|---|---|---|
| BBa_K4661000 | Coding | HFBI_E. coli_optimized |
| BBa_K4661001 | Coding | HFBI_mutated, E. coli |
| BBa_K4661002 | Coding | HFBII coding sequence, E. coli |
| BBa_K4661003 | Coding | HFBII, E. coli codon optimized |
| BBa_K4661004 | Coding | HFBII mutated, E. coli codon optimized |
| BBa_K4661005 | Coding | HGFI, codon optimized for E. coli |
| BBa_K4661006 | Coding | HGFI partially mutated, codon optimized for expression in E. coli |
| BBa_K4661007 | Coding | HGFI mutated, codon optimized for expression in E. coli |
| BBa_K4661009 | Coding | MBSP1, codon optimized for E. coli |
| BBa_K4661010 | Reporter | SGGSGGS linker |
| BBa_K4661011 | Reporter | His-tagged sfGFP |
| BBa_K4661012 | Coding | AcGFP, E. coli optimized |
| BBa_K4661013 | Coding | HFBI-sfGFP fusion |
| BBa_K4661014 | Coding | MBSP1-sfGFP fusion |
| BBa_K4661016 | Coding | HFBI, Yarrowia optimized |
| BBa_K4661017 | Coding | HFBI mutated, Yarrowia optimized |
| BBa_K4661018 | Coding | HFBII, Yarrowia optimized |
| BBa_K4661019 | Coding | HFBII mutated, Yarrowia optimized |
| BBa_K4661020 | Coding | HGFI, Yarrowia optimized |
| BBa_K4661021 | Coding | HGFI partially mutated, Yarrowia optimized |
| BBa_K4661022 | Coding | HGFI mutated, Yarrowia optimized |
| BBa_K4661023 | Coding | MBSP1, Yarrowia optimized |
| BBa_K4661024 | Coding | MATalpha1 signal peptide |
- Cui, L., Cheng, C., Qiu, Y., Jiang, T. & He, B. Excretory overexpression of hydrophobins as multifunctional biosurfactants in E. coli. Int. J. Biol. Macromol. 165, 1296–1302 (2020).
- Gomes, N., Waché, Y., Teixeira, J. A. & Belo, I. Oil-in-water emulsions characterization by laser granulometry and impact on γ-decalactone production in Yarrowia lipolytica. Biotechnol. Lett. 33, 1601–1606 (2011).
- Cheng, Y. et al. Soluble hydrophobin mutants produced in Escherichia coli can self-assemble at various interfaces. J. Colloid Interface Sci. 573, 384–395 (2020).