Engineering

This biobrick contains:

Click on each one of them to learn more about what this biobrick contains.

Constitutive GAP promoter
Drives the continuous expression of both YFP and the bioglue.
fwYellow gene
Encodes Yellow Fluorescent Protein (YFP), the chromoprotein we selected. The sequence has been optimized for expression in S. cerevisiae. The fwYellow gene is a BioBrick created by the Uppsala iGEM team in 2013.
Bioglue genetic sequence
Encodes a bioglue made from a fusion of two natural proteins to significantly enhance adhesion properties: Masp1 from spider silk and Cp19k from barnacles. These proteins have individual adhesion strengths of 22.2 mJ/m² and 2.2 mJ/m², respectively, but their fusion increases this to 39.9 mJ/m² (Ye et al. (2023). The sequences were optimized for expression in S. cerevisiae.
Cellulose Binding Domain (CBD)
Each protein (both the colored and the adhesive) contains a CBD to allow binding to cellulose. The CBD sequence was adapted from Gilbert et al. (2021).
Alpha factor signal peptide
Derived from the MAT alpha mating system, this sequence acts as a signal peptide to facilitate protein secretion. Once secreted, the signal peptide is cleaved, leaving only the mature protein.
P2A system
Enables co-expression of both YFP and the bioglue protein from a single mRNA transcript. This system is optimized for eukaryotic cells and allows simultaneous synthesis of YFP and the adhesive protein in our context.
URA3 gene
Selected as a marker for yeast transformation. This gene allows the URA- yeast strain to synthesize uracil, enabling cells that have successfully integrated the plasmid containing URA3 to grow in uracil-deficient medium.
Homologous sequences (URA5’ and URA3’)
Facilitate plasmid integration into the yeast chromosome via homologous recombination.

1. We started by integrating the URA3-URA3' fragment into Plasmid A, which already contained the URA5' fragment, GAP promoter, alpha factor, fwYellow, and CBD. The result of this construction is Plasmid C. The alpha factor, fwYellow, and CBD genes had to be synthesized, as their sequence were optimized for S. cerevisiae and were not available in any strains we had access to. As a result, we chose to synthesize the entire plasmid. Initially, we aimed to amplify the URA3-URA3' fragment via PCR, but due to the presence of an additional BsaI restriction site in the organism we used for amplification, we decided to synthesize this fragment as well. We used the HiFi Assembly technique to seamlessly join the DNA fragments, ensuring the precise integration of the URA3-URA3' fragment.
2. The fragments containing the P2A sequence, the alpha factor, and the coding sequences of Masp1, Cp19k, and CBD were synthesized, the resulting plasmid is plasmid B.
3. Finally, biobricks from plasmid C and plasmid B were PCR amplified and fused using HiFi assembly resulting in plasmid D.

The conformity of the plasmid was assessed by (i) performing PCR to amplify the fragment of interest (ii) performing a restriction map to validate the assembly and finally (iii) by sequencing to verify the conformity of the final plasmid.

During the process, we identified the presence of an unexpected BsaI site in plasmid A. In hindsight, we realized that if we had carefully checked the sequence of synthesized Plasmid A earlier, we could have identified the extra BsaI site sooner and saved time. Moving forward, we decided to amplify the linear form of our plasmid using PCR instead of relying on restriction enzymes for the next steps.

Once Plasmid D is assembled, it needs to be recombined into S. cerevisiae (BY4741). The homologous regions URA5'and URA3' will facilitate the insertion of the plasmid into the yeast chromosome.

We made S. cerevisiae competent using the Lithium Acetate method. We then transformed the yeast with the 6.3 kbp digestion product of Plasmid D using XhoI. Selection was carried out on a medium lacking uracil since the yeast strain is URA-. If the URA3 gene from the plasmid is successfully integrated into the genome, it will restore the yeast’s ability to synthesize uracil, allowing growth on the selective medium.

First, the correct chromosomal integration of our construct had to be checked. Then, the production of the proteins had to be assayed.
To verify the correct insertion of the plasmid into the yeast genome, we extracted genomic DNA from 8 clones and performed PCR amplification using the primers iGEM1 and iGEM17.

Then we cultured the yeasts to detect the production of the engineered proteins. While we don’t have a way to directly verify the secretion of Masp1-Cp19k-CBD, we can check the secretion of YFP. We anticipated that, according to our design, the fluorescence should be retrieved in the soluble fraction of the cultures as the protein encompass a secreting signal peptide (Alpha factor signal peptide). We recorded the fluorescence of the wt strain and the engineered strain after growth in YPG medium and collection of the pellet fraction as well as the supernatant fraction. However, no significant fluorescence could be measured.

The correct insertion of the genetic construct into the yeast chromosome was demonstrated using colony PCR, we can thus conclude that the strain is conform. However, the test of the phenotype failed. Thus, we designed another test, more sensitive to detect the YFP.

To detect the production of the YFP we decided to perform a western blot using commercial anti-GFP antibodies. These antibodies should recognize YFP, as the sequences of the two proteins only differ from a few amino acids and the 3D structure is the same. To test our hypothesis (production of YFP in the supernatant), we prepared yeast crude extracts, soluble fraction (culture supernatant) and decided to concentrate the soluble fraction to enhance the signal, anticipating that, if produced, the YFP may be present in small amounts.

We confirmed that our construct is functional: YFP is synthesized and secreted as expected. The observed protein sizes match the predicted values, indicating that the P2A system is functioning correctly. Additionally, since YFP is secreted, we can conclude that the alpha factor is also working as intended, facilitating the secretion of the protein. In conclusion, both the P2A system and the alpha factor are effective in our construct, the chromoprotein is produced. We can expect that the bio-adhesive protein is produced as well but didn’t have time to test it.

1. The production of cellulose by K. rhaeticus. In the article (Gilbert et .al, 2021) on which the co-culture system was based, the cellulose-producing microorganism is Komagateibacter rhaeticus. The objective is to verify that K. rhaeticus is indeed a cellulose-producing bacterium.
2. Once the cellulose producing medium is optimized, the conditions leading to a massive production had to be determined.
3. Finally, to package the cellulose it must be dried and sterilized

1. K. rhaeticus was cultured in both YPG and HS media. YPG medium was used as described in the article, while HS medium was also employed, HS medium is optimal for cultivating this type of bacteria according to the manufacturer.
2. Cellulose production is a process that quickly depletes the co-culture medium. The cellulose patches produced from a co-culture are placed in fresh culture after 3 days to prevent production from being halted by medium depletion.
3. In the foundational article, cellulose is dried between two sheets of absorbent paper and two sheets of parafilm. This procedure was replicated in our experiments. To attempt to sterilize the cellulose patches, we used UV irradiation.

Following the various cultures in different media, it was observed that both YPG and HS media support cellulose production.

Then, we aimed at proving that the material observed in YPG is cellulose. YPG medium and YPG medium supplemented with 2% cellulase were inoculated with K. rhaeticus.

The nature of the material present in the medium is concluded from visual observation. In the medium without cellulase, cellulose flakes are observed, while no material is present in the medium with cellulase.
To enhance the production, the cellulose patches produced from a co-culture are placed in fresh culture after 3 days to prevent production from being halted by medium depletion. However, the cellulose is not thicker after several passages.
The cellulose was dried between two sheets of absorbent paper and two sheets of parafilm.
Finally, the cellulose patches were placed in a UV box for 30 minutes. Following this, the cellulose patches were digested and diluted. These solutions were spread on solid YPG medium, and the plates were incubated for 2 days at 30°C. Regardless of the media tested, the cellulose patches are not sterile.

The results obtained in the article were successfully reproduced, as K. rhaeticus produced cellulose in YPG medium. K. rhaeticus also produced cellulose in HS medium. The results for cultures in HS and YPG media were similar. Given that YPG medium was used in the article, it was selected for the remaining experiments. The observed material is indeed cellulose, as the addition of cellulase to the medium results in no observable material. The cellulose is not thicker after several passages. A continuous culture system (fed-batch) should be considered after establishing the appropriate medium conditions. We succeeded in drying the cellulose between two sheets of paper. Finally, the sterilization procedure we used was not effective enough. A different sterilization technique for the cellulose patches needs to be developed. We are in a situation where the cellulose patch is not functional; if adherent and colored proteins are fixed to the cellulose, sterilization methods applied to the cellulose membrane alone should be tested, as these methods may not be effective on functional patches.