Part:BBa_K5143024
Yellow protein and BioGlue protein secreted by Saccharomyces cerevisiae
Description
This composite part corresponds to the association of two composite parts (BBa_K5143023 & BBa_K5143022) linked by a P2A system (BBa_K5143012), all under the control of a single promoter. During translation, this would give two distinct fusion proteins from the same transcriptional unit, which would then be secreted by their respective alpha factors and associate with the cellulose membrane via their CBD domains. This composite part is cloned within the backbone (BBa_K5143005) and resulting in the following integrative plasmid: BBa_K5143025 Then it is digested by XhoI so that the composite part is framed by zones of homology. Following transformation, the whole is integrated by homologous recombination within the yeast Saccharomyces cerevisiae .
Figure 1: Schematic diagram of the bioglue genetic construct linked to chromoprotein yellow by the P2A peptide sequence, flanked by the GAP promoter and TDH1 terminator.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1361
Illegal PstI site found at 2789
Illegal PstI site found at 3092
Illegal PstI site found at 3185
Illegal PstI site found at 3191 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1361
Illegal PstI site found at 2789
Illegal PstI site found at 3092
Illegal PstI site found at 3185
Illegal PstI site found at 3191 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1361
Illegal BamHI site found at 1906
Illegal BamHI site found at 2620 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1361
Illegal PstI site found at 2789
Illegal PstI site found at 3092
Illegal PstI site found at 3185
Illegal PstI site found at 3191 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1361
Illegal PstI site found at 2789
Illegal PstI site found at 3092
Illegal PstI site found at 3185
Illegal PstI site found at 3191
Illegal NgoMIV site found at 1876
Illegal AgeI site found at 1915 - 1000COMPATIBLE WITH RFC[1000]
Construction
The composite biobricks were optimised for transcription and synthesis in S. cerevisiae .
This composite part was first synthesized to obtain the "yellow" fragment in the backbone (BBa_K5143005). The newly plasmid was checked by sequencing, in order to carry out a new cloning to add the "bioglue" fragment. The plasmid has been opened by PCR and the fragment has been cloned by HIFI to obtain the final construct BBa_K5143025.
This construction will be carried out without scarring.
Size: 6365 bp
References
1) A bioinspired synthetic fused protein adhesive from barnacle cement and spider dragline for potential biomedical materials - PubMed.
2) Gilbert, C. et al. Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat Mater 20, 691–700 (2021).
3) A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in Saccharomyces cerevisiae | ACS Synthetic Biology
4) Liljeruhm, J. et al. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. Journal of Biological Engineering 12, 8 (2018).
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