Difference between revisions of "Part:BBa K5143025"
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<h1>Description</h1> | <h1>Description</h1> | ||
<p> | <p> | ||
| − | This part was designed to be used in <i> Saccharomyces cerevisiae </i>. Its main components are <i> fwYellow </i> (<a href="https://parts.igem.org/Part:BBa_K5143023">BBa_K5143023</a>) and <i> Cp19k-MaSp1 </i> | + | Our team has nicknamed this plasmid “<b>plasmid D</b>”. This part was designed to be used in <i> Saccharomyces cerevisiae </i>. Its main components are <i> fwYellow </i> (<a href="https://parts.igem.org/Part:BBa_K5143023">BBa_K5143023</a>) and <i> Cp19k-MaSp1 </i> |
(<a href="https://parts.igem.org/Part:BBa_K5143022">BBa_K5143022</a>) fused together (<a href="https://parts.igem.org/Part:BBa_K5143024">BBa_K5143024</a>). <br> | (<a href="https://parts.igem.org/Part:BBa_K5143022">BBa_K5143022</a>) fused together (<a href="https://parts.igem.org/Part:BBa_K5143024">BBa_K5143024</a>). <br> | ||
By digesting this part with XhoI restriction enzyme, the linearized fragment could be transformed into the yeast in order to recombinate with the Ura locus in <i> S. cerevisiae </i> BY4741 strain. Then, the yeast will express the alphafactor-fwYellow-CBD-P2A-alphafactor-MaSp1-CBD gene. P2A (<a href="https://parts.igem.org/Part:BBa_K5143012">BBa_K5143012</a>) system enables the ribosomal-switch on the mRNA, and leads to the formation of two different proteins : alphafactor-fwYellow-CBD and alphafactor-Cp19k_MaSp1-CBD.<br> | By digesting this part with XhoI restriction enzyme, the linearized fragment could be transformed into the yeast in order to recombinate with the Ura locus in <i> S. cerevisiae </i> BY4741 strain. Then, the yeast will express the alphafactor-fwYellow-CBD-P2A-alphafactor-MaSp1-CBD gene. P2A (<a href="https://parts.igem.org/Part:BBa_K5143012">BBa_K5143012</a>) system enables the ribosomal-switch on the mRNA, and leads to the formation of two different proteins : alphafactor-fwYellow-CBD and alphafactor-Cp19k_MaSp1-CBD.<br> | ||
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</p> | </p> | ||
<img src="https://static.igem.wiki/teams/5143/bba-k5143025-functionalised-cellulose.png" width="800" alt="Cp19k-MaSp1"> | <img src="https://static.igem.wiki/teams/5143/bba-k5143025-functionalised-cellulose.png" width="800" alt="Cp19k-MaSp1"> | ||
| − | <figcaption>Figure 1: Cellulose functionalization process</figcaption> | + | <figcaption><i><u>Figure 1:</u> Cellulose functionalization process</i></figcaption> |
<h1>Construction</h1> | <h1>Construction</h1> | ||
<p> | <p> | ||
The codons were optimised for synthesis and expression in <I> Saccharomyces cerevisiae </I>. <br> | The codons were optimised for synthesis and expression in <I> Saccharomyces cerevisiae </I>. <br> | ||
| − | + | The pUC57 backbone was synthesized with the alphafactor-fwYellow-CBD sequence in it. Then, this plasmid was linearised by PCR in order to clone the alphafactor-Bioglue-CBD in it. The alphafactor-Bioglue-CBD had been previously synthesized. Afterwards, plasmids obtained in clones has been sequenced in order to verify the insertion of the Bioglue fragment in the plasmid. <br> | |
| − | This composite part is | + | <br> |
| − | + | This composite part is composed of the following parts:<br> | |
| − | + | - the GAPpromotor-alphafactor-fwYellow-CBD-P2A-alphafactor-Cp19k_MaSp1-CBD <a href="https://parts.igem.org/Part:BBa_K5143024">BBa_K5143024</a> <br> | |
| − | + | - the pUC57 backbone <a href="https://parts.igem.org/Part:BBa_K5143005">BBa_K5143005</a> | |
| − | + | ||
| − | + | ||
</p> | </p> | ||
| + | |||
</body> | </body> | ||
</html> | </html> | ||
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<partinfo>BBa_K5143025 SequenceAndFeatures</partinfo> | <partinfo>BBa_K5143025 SequenceAndFeatures</partinfo> | ||
| − | + | <html> | |
| + | <body> | ||
| + | <h1>References</h1> | ||
| + | <p> | ||
| + | (1) Ye L, Liu X, Li K, Li X, Zhu J, Yang S, Xu L, Yang M, Yan Y, Yan J. A bioinspired synthetic fused protein adhesive from barnacle cement and spider dragline for potential biomedical materials. Int J Biol Macromol. 2023 Dec 31;253(Pt 5):127125. doi: <a href="https://doi.org/10.1016/j.ijbiomac.2023.127125" target="_blank">10.1016/j.ijbiomac.2023.127125</a>. Epub 2023 Sep 28. PMID: 37776922. <br> | ||
| + | <br> | ||
| + | (2) Gilbert, C. et al. Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat Mater 20, 691–700 (2021). | ||
| + | A Yeast Modular Cloning (MoClo) Toolkit Expansion for Optimization of Heterologous Protein Secretion and Surface Display in <i>Saccharomyces cerevisiae</i> | ACS Synthetic Biology. https://pubs.acs.org/doi/10.1021/acssynbio.3c00743. <br> | ||
| + | <br> | ||
| + | (3) Liljeruhm, J. et al. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. Journal of Biological Engineering 12, 8 (2018) <br> | ||
| + | <br> | ||
| + | (4) Liu, Z. et al. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep 7, 2193 (2017). Mukherjee, M. & Wang, Z. Q. A well-characterized polycistronic-like gene expression system in yeast. Biotechnology and Bioengineering 120, 260–271 (2023). <br> | ||
| + | <br> | ||
| + | (5) Müntjes, K. et al. Establishing Polycistronic Expression in the Model Microorganism Ustilago maydis. Front Microbiol 11, 1384 (2020). | ||
| + | </p> | ||
| + | </body> | ||
| + | </html> | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K5143025 parameters</partinfo> | <partinfo>BBa_K5143025 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
Latest revision as of 16:17, 1 October 2024
Plasmid D
Description
Our team has nicknamed this plasmid “plasmid D”. This part was designed to be used in Saccharomyces cerevisiae . Its main components are fwYellow (BBa_K5143023) and Cp19k-MaSp1
(BBa_K5143022) fused together (BBa_K5143024).
By digesting this part with XhoI restriction enzyme, the linearized fragment could be transformed into the yeast in order to recombinate with the Ura locus in S. cerevisiae BY4741 strain. Then, the yeast will express the alphafactor-fwYellow-CBD-P2A-alphafactor-MaSp1-CBD gene. P2A (BBa_K5143012) system enables the ribosomal-switch on the mRNA, and leads to the formation of two different proteins : alphafactor-fwYellow-CBD and alphafactor-Cp19k_MaSp1-CBD.
In our project, these two proteins will be secreted by the yeast and will bind to the cellulose (thanks to the fused CBD) in order to functionalize the cellulose.
Construction
The codons were optimised for synthesis and expression in Saccharomyces cerevisiae .
The pUC57 backbone was synthesized with the alphafactor-fwYellow-CBD sequence in it. Then, this plasmid was linearised by PCR in order to clone the alphafactor-Bioglue-CBD in it. The alphafactor-Bioglue-CBD had been previously synthesized. Afterwards, plasmids obtained in clones has been sequenced in order to verify the insertion of the Bioglue fragment in the plasmid.
This composite part is composed of the following parts:
- the GAPpromotor-alphafactor-fwYellow-CBD-P2A-alphafactor-Cp19k_MaSp1-CBD BBa_K5143024
- the pUC57 backbone BBa_K5143005