Part:BBa_K5143025

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.

Figure 1: Cellulose functionalization process

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

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal suffix found in sequence at 3567
  Illegal EcoRI site found at 1361
  Illegal EcoRI site found at 7006
  Illegal EcoRI site found at 7533
  Illegal PstI site found at 2789
  Illegal PstI site found at 3092
  Illegal PstI site found at 3185
  Illegal PstI site found at 3191
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1361
  Illegal EcoRI site found at 7006
  Illegal EcoRI site found at 7533
  Illegal SpeI site found at 3568
  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 PstI site found at 3582
  Illegal NotI site found at 3575
  Illegal NotI site found at 7539
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1361
  Illegal EcoRI site found at 7006
  Illegal EcoRI site found at 7533
  Illegal BamHI site found at 1906
  Illegal BamHI site found at 2620
  Illegal XhoI site found at 5171
  Illegal XhoI site found at 7012
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal suffix found in sequence at 3568
  Illegal EcoRI site found at 1361
  Illegal EcoRI site found at 7006
  Illegal EcoRI site found at 7533
  Illegal PstI site found at 2789
  Illegal PstI site found at 3092
  Illegal PstI site found at 3185
  Illegal PstI site found at 3191
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 7533
  Illegal EcoRI site found at 1361
  Illegal EcoRI site found at 7006
  Illegal SpeI site found at 3568
  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 PstI site found at 3582
  Illegal NgoMIV site found at 1876
  Illegal AgeI site found at 1915
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 4280
  

References

(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: 10.1016/j.ijbiomac.2023.127125. Epub 2023 Sep 28. PMID: 37776922.

(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 Saccharomyces cerevisiae | ACS Synthetic Biology. https://pubs.acs.org/doi/10.1021/acssynbio.3c00743.

(3) Liljeruhm, J. et al. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. Journal of Biological Engineering 12, 8 (2018)

(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).

(5) Müntjes, K. et al. Establishing Polycistronic Expression in the Model Microorganism Ustilago maydis. Front Microbiol 11, 1384 (2020).