We designed three plasmids to achieve the purpose of introducing the ion channel "NAN-IAV mode" with Tether to Saccharomyces cerevisiae BY4741. We used the yeast expression vector pESC-HIS as the skeleton, introduced the DmNan gene module, and constructed the pADH1 plasmid. Using the expression vector pESC-LEU in yeast as the skeleton, the DmIav and SunTag gene modules were introduced to construct the pTDH3p plasmid. Using the yeast expression vector pESC-URA as the skeleton, the scFV and actin gene modules were introduced to construct the pACT1p plasmid. In the final digestion verification, linearization was performed by Xho I digestion with a band length of 12534bp, EcoR I digestion plasmid to 3970bp and 8564bp, and EcoR V digestion to 7367bp and 5167bp, verifying the correctness of pTDH3p. Linearization by Xho I digestion with a band of 9149 bp; HindIII. digestion bands are 2991 bp and 6158 bp; Sal I digestion bands are 3031 bp and 6118 bp. The correctness of pACT1p was verified.

Plasmid
Figure 1, and the pADH1 plasmid


Figure 2, the pTDH3p plasmid

Figure 3, pTDH3p plasmid digestion and validation

Figure 4, the pACT1p plasmid

Figure 5, Verification of pACT1p plasmid digestion

Our experiment did not reach the stage of physiological and biochemical experiments due to objective reasons, but we made a hypothesis for this. In our future experimental program, we will perform physiological and biochemical stimulation on yeasts containing the "NAN-IAV pattern" and the Tether structure, and explore the frequency and loudness of the sound that our yeast can perceive. The data and conclusions drawn from this will verify our conjectures and assumptions about yeast's sound perception. While it is a great pity that we were unable to publish this part, we believe we have taken the first step in our exploration of microbial sound perception.