Revision as of 14:06, 1 October 2024

Yellow protein and BioGlue protein secreted by Saccharomyces cerevisiae <style>

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Description

This composite part was designed and conceived for use in the yeast Saccharomyces cerevisiae to develop the BIO Snare project. The goal of this project is to functionalize the cellulose produced by the bacterium Komagataeibacter rhaeticus using recombinant proteins produced by the yeast S. cerevisiae. Le projet est expliqué plus précisément sur : Description Thus, this composite part corresponds to the association of two other composite parts: the recombinant chromoprotein, fwYellow (BBa_K5143023), and the recombinant bioglue (BBa_K5143022). They are linked by a P2A system (BBa_K5143012), and the entire construct is under the control of a single promoter.

It is well known that polycistronic messenger RNAs do not exist in eukaryotic cells. However, this construction, using the P2A system, allows for the post-translational retrieval of two distinct fusion proteins from the same transcriptional unit. These proteins are subsequently secreted from the cell using their respective α-factors and associated with the cellulose membrane via their CBD domains. To integrate this fragment into the yeast chromosome, the composite part is cloned into the backbone (BBa_K5143005), resulting in the following integrative plasmid: BBa_K5143025, referred to as plasmid D. It is then digested with XhoI so that the composite part is flanked by regions of homology specific to the locus of the URA3 gene, allowing for the selection of transformants.

Following transformation, the entire construct is integrated by homologous recombination into the yeast Saccharomyces cerevisiae. It is then co-cultured with the bacterium K. rhaeticus to produce functionalized cellulose patches.

Sequence and features

Construction

The composite BioBricks were optimized for transcription and translation in Saccharomyces cerevisiae. The construction of this composite part was carried out in several steps. Due to economic reasons, it was split into two parts, which were placed in plasmids and synthesized separately. Steps in the construction of composite part BBa_K5143024: (for more details see: Engineering)

- PCR amplification of the recombinant YFP protein sequence BBa_K5143023 from the synthesized plasmid pUC57-A
- PCR amplification of the recombinant bioglue protein BBa_K5143022 + P2A system from the plasmid pUC57-B
- Linearization by PCR of the plasmid pUC57 (synthesized with the URA3 homology regions and the URA3 gene), giving the plasmid backbone BBa_K5143005
- HiFi cloning (NEBuilder HIFI DNA Assembly Cloning kit, ref: E5520S) of the backbone BBa_K5143005 with the recombinant YFP protein sequence BBa_K5143023, yielding the plasmid pUC57-C
- Transformation into E. coli DH5α
- After verification by colony PCR and restriction mapping, sequencing was performed to ensure that this first intermediate construct was correct
- Linearization by PCR of the plasmid pUC57-C followed by HiFi cloning with BBa_K5143022, yielding the plasmid pUC57-D BBa_K5143025
- Transformation into E. coli DH5α
- Verification by colony PCR and restriction mapping; sent for sequencing
- Digestion of plasmid pUC57-D BBa_K5143025 with the restriction enzyme XhoI to release the composite part BBa_K5143024, including the homology regions and the URA3 gene, using the XhoI restriction enzyme

Size of composite part BBa_K5143024: 4229 bp
Size of composite part BBa_K5143024 with homology regions and yeast selection gene: 6344 bp

Contribution

In order to make this BioBrick accessible to a larger number of users for their projects, we decided not to include the homology regions required for integration into the genome of S. cerevisiae BY4741. This allows you to use this BioBrick in your projects to secrete two proteins from a single mRNA in a eukaryotic organism via the P2A system. The only limitation is that the organism must support secretion through the α-factor system (typically yeast, such as Saccharomyces or Pichia). Remember to optimize the sequences for the organism you are using, as ours are optimized for S. cerevisiae. If you wish to modify S. cerevisiae for your own purposes, feel free to use our backbone plasmid BBa_K5143005, which allows for the integration of heterologous genes into its genome!