Parts

Overview

The parts below are inspired from Ye, L. et al.¹, Gilbert, C. et al.², Liu, Z. et al.³, Brake, A. J.et al.⁴, Liljeruhm, J. et al.⁵. Together, these parts enable the functionalization of bacterial cellulose by BY4741 Saccharomyces cerevisiae strain.

The sequences of initial basic parts from the literature were optimized for the expression and the synthesis in S. cerevisiae, it’s why all of the parts we used were synthesized.

This page provides comprehensive information regarding the basic parts, composite parts and plasmid parts designed by the UnivLyon1-INSALyon team and used in the BIO Snare project. Should further information be required regarding the nature and utilization of basic, composite and plasmid parts, the UnivLyon1-INSALyon team kindly request that you contact us at igemlyon1.insa2024@gmail.com e-mail address.

Basic parts

Name	Nickname	Description	Length	RFC
BBa_K5143007	ScCBD_cex'V1	The cellulose binding domain protein binds permanently to cellulose. In our project, we fuse it with our recombinant proteins, produced by S. cerevisiae, so that they associate with the cellulose produced by K. rhaeticus in co-culture with the yeast.	336 bp	10, 12, 23, 1000
BBa_K5143008	ScCBD_cex'V2	The sequence of this cellulose binding domain protein has been modified to avoid homologous recombination once integrated into the S. cerevisiae genome.	336 bp	10, 12, 21, 23, 25, 1000
BBa_K5143009	alpha factor secretion signal V1	Protein secretion signal in yeast derived from the S. cerevisiae Matα pheromone system. The sequence is cleaved during protein excretion.	255 bp (2 segments)	10, 12, 21, 23, 25, 1000
BBa_K5143010	alpha factor secretion signal V2	Protein secretion signal in yeast, which has been modified to prevent homologous recombination once integrated into the S. cerevisiae genome.	255 bp (2 segments)	10, 21, 23, 25, 1000
BBa K5143020	AGA2	The AGA2 gene encodes a small subunit involved in the surface display system in S. cerevisiae, allowing the attachment of recombinant proteins to the cell wall. It is commonly used to display proteins of interest on the surface of yeast cells.	50 bp	10, 12, 23, 1000
BBa_K5143012	P2A	The P2A peptide is a short protein motif that enables the co-expression of multiple proteins from a single mRNA transcript by facilitating a "skip" in translation. It allows the production of distinct proteins while leaving a small amino acid residue at the C-terminus of the first protein. This allows the creation of polycistronic mRNAs in eukaryotes.	57 bp	10, 12, 21, 23, 25, 1000
BBa_K5143004	GS linker	The GS linker is a flexible peptide sequence that provides spatial and functional flexibility between two fused proteins, enhancing their activity and preventing steric hindrance.	36 bp	10, 12, 21, 23, 25, 1000
BBa_K5143001	MaSp1	Spider silk adhesive protein (from Nephila clavata).	744 bp	1000
BBa_K5143002	cp19k	Barnacle adhesive protein (from Megabalanus rosa).	519 bp	10, 12, 21, 23, 25, 1000
BBa_K5143006	fwYellow	The fwYellow gene is a variant of the green fluorescent protein (GFP) gene that we use here as a colored chromoprotein, inspired by the work done by the iGEM team Uppsala in 2013.	708 bp	100
BBa K5143014	mRuby2	Red fluorescent protein optimized for S. cerevisiae, which allows us to test the activation of the ADH1 promoter.	708 bp	10, 12, 21, 23, 25, 1000
BBa_K5143015	venus	Yellow fluorescent protein, optimized for S. cerevisiae and derived from YFP (Yellow Fluorescent Protein), allows us to test the activation of the GAP promoter.	714 bp	10, 12, 21, 23, 25, 1000

Composites parts

Name	Nickname	Description	Length	RFC
BBa_K5143023	recombinant fwYellow	This recombinant protein, once translated, can be secreted thanks to its alpha factor secretion signal domain and attached to cellulose through its ScCBD_cex'V1 domain. This will allow the cellulose to be stained yellow.	1313 bp	1000
BBa_K5143003	bioglue	Cp19k_MaSp1 bioglue composite part is a fusion between two bioglues genes to improve their stickness. A linker between the two bioglues gene allows the good conformation of the fusion protein	1299 bp	1000
BBa_K5143022	recombinant bioglue	This composite part is composed of the Cp19k_MaSp1 bioglue fused to a CBD_V2 at 3'extremity that allows the bioglue to bind to the cellulose, and also fused to an alphafactor secretion signal V2 at 5'extremity to enable the secretion by the S. cerevisiae yeast of the fused protein.	1890 bp	1000
BBa_K5143024	recombinant bioglue_fwYellow	This composite part corresponds to the association of two composite parts linked by a P2A system, 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.	3988 bp	1000
BBa_K5143021	recombinant mRuby	This composite part is the fusion of the mRuby2 fluorescent protein gene with the AGA2 secretion system gene. Additionally, a 6xHis tag is included, along with the ADH1 promoter and the TDH1 terminator. It is ready to be directly cloned into an expression vector.	1733 bp	10, 12, 21, 23
BBa_K5143026	recombinant Venus	This composite part is the fusion of the Venus fluorescent protein gene with the alpha factor v1 secretion system gene. Additionally, the GAP promoter and the ENO1 terminator are added to both ends of the sequence of this recombinant protein to be directly cloned into an expression vector.	1887 bp	10, 12, 23, 1000

Plasmids

Name	Nickname	Description	Length	RFC
BBa K5143005	plasmid backbone	This backbone is used to clone various genetic elements with the aim of amplifying them in E. coli DH5 alpha, and then releasing the recombinant cassette of interest to be integrated into the V chromosome of Saccharomyces cerevisiae BY4741.	3945 bp	/
BBa_K5143025	Plasmid D	Plasmid containing the final construct with the fwYellow_bioglue sequence, ready to be digested by the XhoI restriction enzyme for integration into the S. cerevisiae genome.	8206 bp	/
BBa_K5143027	Plasmid Venus/mRuby	Plasmid containing the sequences of two recombinant proteins, Venus and mRuby, which allow testing the efficiency of two strong constitutive promoters, as well as two secretion systems: alpha factor and AGA2. This plasmid also serves as a backup if the P2A system does not work (in which case the CBD domains will need to be added).	7597 bp	/

References

[1] Ye, L. et al. (2023). A bioinspired synthetic fused protein adhesive from barnacle cement and spider dragline for potential biomedical materials. International Journal of Biological Macromolecules 253, 127125.

[2] Gilbert, C. et al. (2021). Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat. Mater. 20, 691–700.

[3] Liu, Z. et al. (2017).Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7, 2193.

[4] Brake, A. J. et al. a-Factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae.

[5] Liljeruhm, J. et al. (2018). Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. J Biol Eng 12, 8.

[6] Ma, C. et al. (2021). Ultra-strong bio-glue from genetically engineered polypeptides. Nat Commun 12, 3613.