Part:BBa_K5143021

Ruby fused with AGA2 under the control of the ADH1 promoter

Description

mRuby2 can be used as a reporter system. Here, mRuby2 was used as a transcriptional reporter, to control the transcriptional efficiency of the yeast promoter pADH1 and the efficiency of the AGA2 secretion system. The excitation wavelength is 559nm and the emission wavelength is 600nm.
In yeast, we have tested several promoters to identify the most optimal one, i.e., the one that expresses the highest amount of protein. This BioBrick allows us to evaluate the strong, constitutive ADH1 promoter.

Figure 1. The fluorescence of mRuby is assessed in either the supernatant or within the cells to determine whether the protein is produced and secreted.

Details of the System Function

By quantifying the fluorescence emitted by the cells, we can assess the strength of the promoter, which is crucial in our case to maximize the amount of secreted protein. We have constructed a similar setup using a different strong promoter and another fluorescent protein to compare the promoters. Using the AGA2 pre-peptide, we can determine whether the protein is effectively secreted into the extracellular medium. To achieve this, we will measure the fluorescence in the culture supernatant and verify whether proteins have been produced and secreted. The His tag will enable us to perform Western blots to confirm our results.

Construction

The gene encoding the chimeric mRuby2 protein fused to AGA2 and 6HIS-Tag has been optimized for synthesis and expression in Saccharomyces cerevisiae. We have placed this coding region downstream of the ADH1promoter to ensure strong constitutive expression. The entire construct has been synthesized.
This genetic construct has been cloned into the following plasmid backbone: BBa_K5143005
Resulting in the following integrative plasmid: venus/mRuby plasmid

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 180
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 830
  

References

1. Lam, A. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9, 1005–1012 (2012).

2. Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol. 4, 975–986 (2015).