[1] Design improvement

1、P2A

P2A is a self-shearing peptide, and the 2A peptide is not completely "self-shearing", but plays a role by causing the ribosome to skip the synthesis of glycine and proline peptide bonds at the C-terminal of the 2A element, ultimately leading to the separation of the end of the 2A sequence and the downstream products. After fusion protein expression, P2A can achieve the purpose of isolating fluorescent protein from target protein.

2、Linker choice

For the plasmid pTDH3p, we used 5x (GS) Linker to connect DmIav to SunTag. 5x (GS) Linker is a flexible amino acid with a stretchable space to ensure that the anterior and posterior proteins are not affected. For the plasmid pACT1p,we used 5x (GS) Linker to connect ATC1 to sfGFP for these reasons. In addition, studies have shown that adjusting the nucleotide composition of the Linker sequence (GGGGS)3 can significantly increase the mRNA level of the recombinant fusion protein by a factor of 30 and the transfection efficiency is higher. So we used 3x (GGGGS) Linker to connect scFv and sfGFP.


3、Tether（SunTag-scFv)


SunTag is essentially a set of molecular hooks that can attach multiple copies of bioactive molecules to protein scaffolds that can be used to target some genes or other molecules. Compared to assembled molecules without these hooks, the molecular bioactivity of SunTag is significantly magnified. We plan to use SunTag signaling to amplify downstream signals onto the cytoskeleton to achieve a transition from mechanical stimulation to physiological activity, observe yeast cells' response to sound waves, changes in gene levels, protein levels, and metabolic and growth phenotypes, and explore the life phenomenon of biological perception of sound. The SunTag was developed by researchers in Dr. Ron Vale's lab, a professor of molecular and cellular pharmacology at the University of California, San Francisco and a researcher at the Howard Hughes Institute for Medical Research (HHMI).

[2] Experimental improvement

*Measurement of OD value

Our initial experiment used a small Erlenmeyer flask to culture the bacteria. In order to stagger the measurement time, for BY4741, BY4741-ACT1, and BY4741-DmIav, we connected three strains into 7 bottles, and then divided 21 vials into nine batches for measurement, the first three times using the same medium, and the last six times divided into different media to measure. That is, at the time points of 0, 4, 8, 12, 24, 36, 48, 60, 72 hours, their OD values are measured to detect their population density. To ensure the accuracy of the experiment, we take 2 ml of the bacterium solution 3 times in each bottle separately and take its average value. However, due to the failure of the experimental instrument spectrophotometer instrument and the reason for the culture, the od curve of the bacteria is not completely regular, showing a tortuous rising state. Later we improved the experiment to re-measure. For BY4741, BY4741-ACT1, and BY4741-DmIav, we re-cultured three strains in nine large Erlenmeyer flasks, re-engineered the od curve, and tested their growth. After our improvements, the results of the second test were relatively successful, and we showed it in our "experimental results".