Part:BBa_K5143024

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 produced by the yeast S. cerevisiae. he project is detailed further in: 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.

Figure 1: Descriptive schematic of the composite part BBa_K5143024.

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.

Figure 2: Results of cloning the BioBrick BBa_K514024 into the backbone plasmid BBa_K514005 to obtain plasmid D BBa_K514025.

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.

Figure 3: Schematic representation of the integration of the composite part BBa_K5143024 and the URA3 selection marker into the chromosome of the yeast S. cerevisiae BY4741 to produce the recombinant proteins necessary for the functionalization of cellulose from K. rhaeticus.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Unknown
• 12
  INCOMPATIBLE WITH RFC[12]

  Unknown
• 21
  INCOMPATIBLE WITH RFC[21]

  Unknown
• 23
  INCOMPATIBLE WITH RFC[23]

  Unknown
• 25
  INCOMPATIBLE 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
  
• 1000
  COMPATIBLE WITH RFC[1000]

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!

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 expressed, 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:

Figure 4: Detection of proteins in CE = Crude Extract, S = Supernatant, CS = Concentrated Supernatant. WT: S. cerevisiae BY4741. ScpD1 and ScpD7 : clone 1 and clone 7 of S. cerevisiae transformed with plasmid D.

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 technique
- 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:

Figure 5: Detection of fluorescent proteins. CE = Crude Extract, S = Supernatant and CS = Concentrated Supernatant. A: Positive control: Purified GFP. GFP ctrl: S. cerevisiae GFP-producer. WT: S. cerevisiae BY4741 B: ScpD1 and ScpD7: clone 1 and clone 7 of S. cerevisiae transformed with plasmid D. Expected sizes: alphafactor-YFP-CBD 49.2 kDa, alphafactor-GFP-CBD 48.1 kDa, GFP 27 kDa.

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.

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. Due to time constraints, we were unable to replicate the experiment, but replication will be necessary to confirm these results.

Co-culture between K. rhaeticus and S. cerevisiae

Once the secretion of our proteins was confirmed in clones ScpD1 and ScpD7, we attempted to perform a co-culture between the cellulose-producing bacterium Komagataeibacter rhaeticus and our recombinant yeast strains. For this, a specific medium was inoculated with K. rhaeticus and each of the clones, ScpD1 and ScpD7 (see: Experiments)

Although the recombinant yeast colonies did not display a yellow appearance, we aimed to test whether strains ScpD1 and ScpD7 could grow in the presence of K. rhaeticus, and whether we could confer adhesive properties to the cellulose. After following the co-culture protocol, we were able to obtain a cellulose patch:

Figure 6: Cellulose patch derived from a co-culture between K. rhaeticus and S. cerevisiae ScpD7. The cellulose was not functionalized.

The formation of this patch demonstrated that co-culture with the ScpD1 and ScpD7 strains is possible. However, we were unable to functionalize the cellulose by imparting it with colored or adhesive properties.

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 expression, production, and secretion of the α-factor–YFP–CBD fusion protein demonstrates the effectiveness of Plasmid D in integrating heterologous genes into S. cerevisiae yeast.

Perspectives

To confirm the results observed in the Western blot using anti-GFP antibodies, this manipulation needs to be repeated. In this study, we were unable to determine the expression, production, and secretion of the α-factor–BioGlue–CBD fusion protein because we did not have the necessary antibodies available. We could utilize anti-CBD antibodies to detect our two proteins of interest: α-factor–YFP–CBD and α-factor–BioGlue–CBD. This would allow us to confirm the presence of each protein in the supernatant and further validate the P2A and α-factor systems. To impart colored characteristics to cellulose, it will be necessary to identify new functional chromoproteins in yeast. To address the issue of low protein production, one possible solution would be to use a stronger promoter to drive higher levels of gene expression. Replacing the current promoter with a well-characterized, high-strength promoter, such as the PGK1 or TEF1 promoters, could enhance the transcription of the fusion protein. This would likely increase protein production, making it easier to detect and evaluate its functionality in yeast.

To confirm the effectiveness of our BioGlue, adhesion tests on cellulose should be conducted using a dynamometer.