1. Validation of pTDH3p and pACT1p target plasmids by digestion

(1) pACT1p digestion validation

We selected Xho I enzyme digestion for linearization, with a band of 9149 bp; Hind Ⅲ digestion bands were 2991 bp and 6158 bp; Sal I bands were 3031 bp and 6118 bp.
The results obtained in Figure 1 are consistent with the actual size, so it can be concluded that pACT1p is correctly constructed.



Figure 1 pACT1p plasmid digestion electrophoresis results

(2) pTDH3p digestion validation

We selected Xho I enzyme digestion for linearization, and the strip length is 12534bp, EcoR I enzyme digestion plasmid is 3970bp and 8564bp, EcoR V enzyme digestion is 7367bp and 5167bp. The result in Figure 2 is consistent with the actual size, so it can be concluded that the pTDH3p is correctly constructed.



Figure 2 pTDH3p plasmid digestion electrophoresis results

2. Electrophoretic verification of pTDH3p and pACT1p transformants

(1) Electrophoretic verification of BY4741-ACT1 transformant
We transferred pACT1p plasmid into BY4741 for culture, and selected single colony for colony PCR, as shown in Figure 3. We selected Marker3 for agarose gel validation. As shown in Figure 4, among the 10 swimming lanes, except for the sixth lane, the rest of the bands are 2000 bp, which is close to the length of the pACT1p primer band of 1721 bp. It indicates that the transformation of BY4741 DmNan transformant is successful and can stably exist in the strain.



Figure 3 (electrophoretic verification of BY4741-ACT1 transformant) Figure 4 (BY4741-ACT1 conversion sub plate)



(2) Electrophoretic verification of BY4741-DmIav transformant

We also transferred the pTDH3p plasmid into BY4741 for culture, selected single colony for colony PCR, and selected Marker3 for agarose gel validation. As shown in Figure 5, among the 10 swimming lanes, the bands of the second, third, fifth, sixth, seventh, ninth and tenth swimming lanes are all 2000 bp, which is close to the primer band length of pTDH3p of 2366 bp. It indicated that the transformation of BY4741 DmIav transformant was successful and could stably exist in the strain.



Figure 5 (electrophoretic verification of BY4741 DmIav transformant)

3. Absorbance measurement and growth curve of BY4741, BY4741-DmIav, BY4741-ACT1 strains

We took single colonies of BY4741, BY4741-DmIav and BY4741-ACT1 into the liquid SD medium, expanded and cultured at 29 ℃ at 180rpm until the OD660 value was 0.2, and then inoculated the bacteria into the separately packed 30ml liquid SD medium with 2% of the amount of bacteria. We will take 3 bottles of each kind of bacteria in each period, and expand and cultivate them at 29 ℃ at 180 rpm. The OD660 value shall be measured by ultraviolet spectrophotometer at 0h, 4h, 8h, 12h, 24h, 36h, 48h, 60h and 72h after inoculation, and the average value shall be taken for three times of each bacterial test. The final result is shown in Figure 6. The three strains all showed "S" type growth, and the growth trend of BY4741-DmIav and BY4741-ACT1 transformants was similar to that of BY4741. It can be seen that a single plasmid has a weak impact on the life activities of BY4741.



Figure 6 （BY4741-ACT1，72h）



Figure 7 (BY4741-ACT1 100x oil microscopic examination)



Figure 8（ BY4741，72h）



Figure 9 (BY4741 100x oil microscopic examination)



Figure 10（BY4741-DmIav 72h）



Figure 11 (BY4741-DmIav oil mirror observation)



Figure 12 (Growth Curve of BY4741, BY4741-DmIav, BY4741-ACT1 Bacteria)

4. Observation on Fluorescent Protein of BY4741-DmIav and BY4741-ACT1 Transformers

(1) Observation on Fluorescent Protein of BY4741-ACT1 Transformer



Figure 13 Mingchang



Figure 14 Fluorescence



Figure 15 Merge

Figures 13, 14 and 15 show the results of fluorescence observation.
It can be seen from the figure that pACT1p plasmid can express sfGFP green protein in yeast, and the yeast structure is intact and grows normally, and the fusion protein is successfully expressed.

(2) Observation on Fluorescent Protein of BY4741 DmIav Transformer




Figure 16 Bright field



)Figure 17 Fluorescence



Figure 18 Merge
Figures 16, 17 and 18 show the results of fluorescence observation.

It can be seen from the figure that pTDH3p plasmid can express eBFP blue protein in yeast, and the yeast structure is intact and grows normally. Most yeast structures are intact, and fusion protein expression is successful.
In the experimental stage, we successfully constructed pTDH3p, pADH1, pACT1p plasmids, and verified the correct construction of pACT1p and pTDH3p plasmids. At the same time, they successfully expressed the fusion protein in BY4741. However, due to objective reasons, the construction period of pADH1 plasmid was relatively long, which led to the delay of our experimental plan. We did not have enough time to successfully transfer the three plasmids into BY4741 to verify the effect of sound. The successful expression of two existing plasmids makes us believe that our idea can be realized.

1. Validation of pADH1 by restriction enzyme digestion, detection of transformant electrophoresis, determination of transformant growth curve and observation of fluorescent protein

2. Transformation and validation of three plasmids pTDH3p, pADH1 and pACT1p

3. Physiological and Biochemical Experiment

(1) Dependent variables: loudness, frequency, voice We imagine using mobile phones to make sounds to stimulate yeast. Use the audio generator of the phonex acoustic module of the mobile phone software to change the frequency of the sound, change the size of the sound emitted by the mobile phone, change the size of the sound, and play different sounds to change the timbre of the sound.

(2) Independent variables: cell size (eyepiece micrometer), fluorescence intensity (protein expression), growth rate For the physiological and biochemical reaction of yeast, we imagine to observe the cell size through eyepiece micrometer, observe the fluorescence intensity with fluorescence microscope, and measure the growth rate with spectrophotometer and draw the growth curve.