Latest revision as of 08:49, 2 October 2024

Yellow protein and BioGlue protein secreted by Saccharomyces cerevisiae

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 secreted by the yeast S. cerevisiae. The project is described in more detail at: Description.
Thus, this composite part is made up of two other composite parts. The first encodes the recombinant chromoprotein fwYellow fused to a CBD domain (BBa_K5143023), while the second encodes the recombinant bioglue, which is also linked to a CBD domain (BBa_K5143022). These two recombinant proteins are connected by a self-cleaving P2A system (BBa_K5143012). The entire construct is under the control of a single promoter.

It is well established that polycistronic mRNAs do not occur in eukaryotic cells. However, by utilizing the P2A system, this construct enables the post-translational generation of two distinct fusion proteins from a single 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 ensure the integration of this fragment into the yeast chromosome, the composite part BBa_K5143024 must be flanked by homology regions with the S. cerevisiae BY4741 genome. To achieve this, we have detailed our cloning steps below (see Construction) to produce Plasmid D, which enabled the integration of genes into S. cerevisiae for the establishment of a heterologous expression system. This process resulted in our backbone plasmid BBa_K5143005.
In plasmid D, the composite part BBa_K5143024 is flanked by the 5'URA and 3'URA regions of the yeast S. cerevisiae.

For the integration of BBa_K5143024 into the yeast genome, the yeast must be transformed with a linear fragment. To achieve this, plasmid D was digested with XhoI, which cuts twice in the plasmid outside the URA flanking regions, generating a linear composite part flanked by regions of homology specific to the URA3 gene locus. This allows for the selection of transformants in which the entire construct is integrated by homologous recombination into the S. cerevisiae genome. Correct integration was confirmed by colony PCR. The correct recombinants were then co-cultured with K. rhaeticus to produce functionalized cellulose patches.

Sequence and Features

Construction

The composite BioBricks were optimized for translation in Saccharomyces cerevisiae. The construction of this composite part was carried out in several steps. Due to the lengths of the parts, the composite part had to be split in two parts to be synthesized and directly cloned in plasmids by our sponsor Genecust.

Steps in the construction of composite part BBa_K5143024: (for more details see: Engineering)

- Linear amplification by PCR of the plasmid plasmid A
- HiFi cloning (NEBuilder HIFI DNA Assembly Cloning kit, ref: E5520S) of the linearized plasmid A with the synthetised fragments corresponding to URA3 homology regions and the URA3 gene, yielding the plasmid plasmid 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
- PCR amplification of the recombinant bioglue protein BBa_K5143022 + P2A system from the plasmid plasmid B
- Linearization by PCR of the plasmid plasmid C followed by HiFi cloning with BBa_K5143022, yielding the plasmid plasmid D BBa_K5143025
- Transformation into E. coli DH5α
- Linearization by PCR of the plasmid plasmid C followed by HiFi cloning with BBa_K5143022, yielding the plasmid plasmid D BBa_K5143025
- Digestion of plasmid plasmid 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

To make this BioBrick more accessible to a wider range of users for their projects, we decided not to include in this composite biobrick 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 the integration of genes into the yeast’s genome in order to create an heterologous expression system!

Composite Part Testing

The previous steps demonstrated that the entire BioBrick was successfully integrated into the genome of S. cerevisiae at the targeted locus. However, successful genomic integration does not necessarily imply protein expression. To assess whether the chromoprotein and the bioglue are produced, and secreted, several tests were conducted. We selected two PCR-positive clones, designated ScpD1 and ScpD7, for which subsequent manipulations were performed.

First, we performed an SDS-PAGE to verify the presence of our two proteins in both clones (ScpD1 and ScpD7) by comparison to the WT strain. Several conditions were tested to determine whether the proteins could be detected in the crude extract (CE), the supernatant (S), and the precipitated supernatant (CS). After Coomassie blue staining, we obtained the following results:

As shown in Figure 4, no additional bands were detected in the crude extract (CE) of both clones compared to the WT, and no bands were observed in the supernatant (S) or the precipitated supernatant (CS). From this figure, two hypotheses can be drawn regarding our proteins:

- they are expressed and produced at levels too low to be detected by this technic
- they are not expressed or produced at all.

To confirm one of our two hypotheses, we performed a Western blot to increase resolution and specifically detect proteins. For the Western blot, we used anti-GFP antibodies, which can also recognize the YFP protein, the product of the fwYellow gene. We reused our two clones, ScpD1 and ScpD7, and applied the same three conditions as before: detection in the crude extract (CE), the supernatant (S), and the precipitated supernatant (CS). Additionally, we included control conditions: the WT strain, as previously, and purified GFP as a positive control. The following results were obtained:

In our BioBrick, YFP is fused to the α-factor and the CBD domain, giving it an expected size of 49.2 kDa on the gel. As shown in Figure 15, no bands are observed in the WT strain, while a band is detected in the purified GFP control under the precipitated supernatant (CS) condition, validating our controls.

For our clones ScpD1 and ScpD7, we observe several bands in the concentrated supernatant, including one band near the expected size of the α-factor–YFP–CBD fusion protein (49.2 kDa). However, other non-specific bands are also observed, although not in the WT condition, suggesting that these bands may correspond to degraded forms of the α-factor–YFP–CBD fusion, where either the α-factor or the CBD could be cleaved.

These results confirm that our two recombinant yeast strains (ScpD1 and ScpD7) appear to express, produce, and secrete the α-factor–YFP–CBD fusion protein of interest. However, the significance of this protein lies in its yellow color, which should be visible to the naked eye. The colonies of ScpD1 and ScpD7 did not exhibit a yellow appearance, suggesting that the protein may be non-functional in yeast or produced at levels too low to be detectable. To determine whether fluorescence would be emitted by the cells, a fluorometric assay was conducted, but no signal was detected. The fusion protein was only identified in the concentrated supernatant using Western blot analysis, a highly specific technique. Due to time constraints, we were unable to replicate the experiment, but replication will be necessary to confirm these results.

Conclusion

In conclusion, we were able to demonstrate the secretion of the α-factor–YFP–CBD fusion protein from our BioBrick via Western blot. The detection of this protein implies several things:

- First, the α-factor appears to function properly, as the protein was detected in the concentrated supernatant of the culture. Therefore, the α-factor can be used to secrete other proteins in yeast.
- Second, the detection of the α-factor–YFP–CBD protein indicates that the innovative P2A system is also functional, allowing the fusion protein to be transcribed independently from the fusion protein corresponding to BioGlue.
- Lastly, the production and secretion of the α-factor–YFP–CBD fusion protein demonstrates the effectiveness of Plasmid D in integrating genes in S. cerevisiae that can perform heterologous expression.

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).