iGEM 2022 — Team bjwz-china

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Notebook

![](https://static.igem.wiki/teams/4203/wiki/notebookkk.jpg)

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Design

Wet lab design

Plasmid design

![](https://static.igem.wiki/teams/4203/wiki/experiment1.jpg)
![](https://static.igem.wiki/teams/4203/wiki/experiment14.jpg)
![](https://static.igem.wiki/teams/4203/wiki/experiment3.jpg)
![](https://static.igem.wiki/teams/4203/wiki/experiment13.jpg)
Plasmid design

pADH1 plasmid

The expression vector pESC-HIS in yeast was used as the skeleton with ADH1t on the skeleton to express the DmNan protein.

![](https://static.igem.wiki/teams/4203/wiki/experiment4.jpg)
pADH1 plasmid

(1)P2A
Self-cleaved polypeptide 2A
Self-cleaved polypeptide 2A was discovered in 1991 in foot-and-mouth disease virus (FMDV), with an average length of 18-22 amino acids, located between the two proteins of the picornavirus family member. The cleavage site of the 2A peptide is generally located between the glycine (G) and the proline (P) at its C terminus. Since cleavage, the amino acid residues at the N end of the 2A peptide connect to the upstream protein, while the proline residues remain connected to the downstream proteins, and usually the 2A peptide residue can be removed by Flynn protease and the signal peptide. The 2A peptide is not fully "self-cleaved" but functions by making the ribosome skip the synthesis of the glycine and proline peptide bonds at the C-end of the 2A element, ultimately leading to the separation of the 2A sequence end and the downstream product.
(2) mCherry
mCherry is a red fluorescent protein from mushroom corals, often marking and tracing certain molecular and cellular fractions.mCherry and most other red fluorescent proteins are proteins isolated from Discosoma species. The advantage of mCherry over other fluorescence is that its color and the most used green fluorescent protein (GFP) enable common labeling, and that mCherry is also highly photostable relative to other monomeric fluorescent proteins.mCherry is quite popular among all of the red fluorescent proteins.

pTDH3p plasmid

The expression vector pESC-LEU was used as the cytoskeleton in yeast to express the DmIav protein.

![](https://static.igem.wiki/teams/4203/wiki/experimen.jpg)
pTDH3p plasmid

(1)SunTag
![](https://static.igem.wiki/teams/4203/wiki/experiment6.jpg)
Suntag

Pros of SunTag compared to traditional fluorescent proteins
• Improved brightness and signal-to-noise ratio
• Less chance of phototoxicity
• Reduced photobleaching
• Simplified, long-term single-molecule tracking

summary
Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a novel protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and in controlling biological outputs.

pACT1p plasmid

The expression vector pESC-URA was used in yeast as the cytoskeleton to express the actin protein.
![](https://static.igem.wiki/teams/4203/wiki/experiment7.jpg)
pACT1p plasmid

(1) scFv
Gene symbol:SCFV
Gene description: single-chain Fv fragment
Gene type: other
Organism: Homo sapiens
Lineage: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo
Annotation information: not in current annotation release
(2)Superfolder GFP
Nine point mutations were integrated: S30R, Y39N, F99S, N105T, Y145F, M153T, V163A, I171V, and A260V. Compared with wild-type GFP, Superfolder GFP has better stability, more efficient folding kinetics (can be folded efficiently at 37 °C), brightness is also higher than EGFP, and exhibits stronger acid and alkali resistance compared to other members of the GFP family.

We designed samples such as the above plasmids to transfer DmNAN and DmIAV gene module into yeast, and used Tether (SunTag-scFv) to amplify downstream signals to the cytoskeleton, realizing the transition from mechanical stimulation to physiological activity, so as to achieve the purpose of the perception of sound by yeast.

Primer design

pTDH3p-f:5→3:CGCTATGATGGGAAATAC pTDH3p-r:5→3:AGGACCACCTTCAACAAC
pACT1-f:5→3:GGAGCCAATGCAGTAATT pACT1-r:5→3:GGAGGTGGTGGTTCTTCT
We designed the primers based on the following principles:
1.Primers were designed in the conserved region of the template cDNA.
2.Primer lengths are generally between 15 and 30 bases.
3.The primer GC content ranged from 40% to 60%. The GC content of the upstream and downstream primers could not differ too much.
4.The 3' end of the primer should avoid the third position of the codon, otherwise the degeneracy is prone, which will affect the specificity and efficiency of the amplification.
5.When the last base at the end of the 3' end of the primer is A, the trigger chain can be synthesized even in the case of mismatch, while when the last chain is T, the initiation efficiency of mismatch is greatly reduced, and that of G and C mismatch is between A and T. Therefore, no A was selected at the 3 ′ -end of the primer.
6.Bases should be randomly distributed.
7. There should be no complementary sequence between the primers themselves, or the primers themselves will fold into a hairpin structure (Hairpin), replicating the primer itself. There should also be no complementarity between the two primers, especially avoiding the overlapping complementarity at the 3 ′ end to prevent the formation of the primer dimer (Dimer and Cross dimer). There can be no consecutive 4 bases complementary between the primers.
8. We analyzed the DG values at different positions with Oligo 6 software, so that the 5 ′ end and intermediate △ G values of the primers should be relatively high, while the 3 ′ end △ G values should be lower.
9.The single strand of the amplified product could not form a secondary structure, and we estimated the stable secondary structure of the mRNA with the RNAstructure prediction, facilitating the selection of the template.

Reagent and instrument

(1)Reagent

1. Plasmid:
pACT1p-scFv-GFP-actin-ADH1t(URA)
pTDH3p-DmIav(CDS)-sunTag-BFP-CYC1t(LEU)
pACT1p-scFv-GFP-actin-ADH1t(URA)
2、bacterial strain:
Feeling BY4741 (10 packs with PEG / LiAc for internal conversion) One portion of BY4741 grown in YNB medium;
3、culture medium
SD-Leu-His-Ura Three deficiency medium: solid powder
PM2171 SD-Leu-His-Ura: Broth
YNB Sterile liquid medium was used at 480 ml
SD Medium was obtained from coolaber Company:PM2171 SD/-His/Leu/Ura Bro identification of product：PM2171－10×0.5L Yeast Media
4、drug
amylaceum : 500g
Uracil: 25g
Uridine: 25g
Histidine: 25g
Methionine: 25g
Leucine: 25g
Four kinds of Nutrition from Solebol Inc
L-His: H0020-25g
L-Met: M0010-25g
L-Leu: L0011-25g
Uracil: U8010-25g
agarose
agal-agal
LB broth
laked blood agar
YPD liquid nutrient medium
Ethylene bromide
Marker(5000bp 15000bp)
Loading buffer
TAE

5、kit

Plasmid small lift kit
Agarose gel DNA recovery kit
Infusion kit
(2)Experimence equipment
microwave oven
electronic balance
A UV-visible spectrophotometer
High pressure sterilization pot
-80°C refrigerator
Water bath pot
Saved boxes
microcentrifuge product model:Mini-4K
TG16A-WS table model high speed centrifuge
metal bath model:OLB-MN
Desktop frozen-type centrifuge/Centrifuge
Gradient PCR instrument
Fully automatic gel image system
Basic electrophoresis generator power supply
Multi-purpose horizontal electrophoresis apparatus
The constant temperature shake bed
Thermostatic incubator
superclean bench
Single-lane pipette(2.5 10 20 100 1000μL)
photon microscope

Computer design

1、Dependent variable: loudness, frequency

We envision sound emitted through cell phones to stimulate yeast. Use the audio generator of the phyphox acoustic module to change the frequency of the sound, change the size of the sound issued by the phone to change the size of the sound, and play different sounds to change the color of the sound.

2、Independent variable: cell size, fluorescence intensity, and growth rate

For the physiological and biochemical responses of yeast, we envisioned cell size by eyescopy, fluorescence intensity, spectrophotometer to measure growth rate and plot growth curves.

3、Data analysis

Data processing was performed using Matlab, which draws the conclusions.

4、Expected test results

With the decibel increase, the yeast response is gradually violent, showing a one-function trend. As the tone increases, the yeast response first intensifies and then weakens, with a quadratic function trend.

Construction stage

1、pADH1 plasmid

optimize(for Saccharomyces cerevisiae(Yeast)) And the genes are routinely synthesized
ADH1p-DmNan(CDS)-P2A-mCherry, add 5' (BamHI) and 3' (SacI),the gene was passed through 5' BamHI and 3' SacI Clone to vector pESC-HIS (Ampicillin),build the plasmid pADH1-DmNan(CDS)-P2A-mCherry-ADH1t,one copy of mini-scale recombinant plasmid DNA was prepared and divided into 1 tube and 1 tube puncture containing this recombinant plasmid.

![](https://static.igem.wiki/teams/4203/wiki/experiment8.jpg)
Optimized pADH1 plasmid

2、pTDH3p plasmid

optimize(for Saccharomyces cerevisiae(Yeast)) The gene TDH3p-DmIav (CDS) -sunTag-BFP was routinely synthesized, supplemented with 5' (SacI) and 3' (XhoI) and cloned into the vector pESC via 5' SacI and 3'XhoI (Ampicillin). The plasmid pTDH3pDmIav (CDS) -sunTag-BFP-CYC1t (LEU) was added to prepare a mini-scale recombinant plasmid DNA and divided into 1 tube and 1 tube containing the recombinant plasmid.

![](https://static.igem.wiki/teams/4203/wiki/experiment9.jpg)
Optimized pTDH3p plasmid

3、pACT1p plasmid

optimize(for Saccharomyces cerevisiae(Yeast)) the gene ACT1p-scFv-GFP-actin, supplemented with 5' (BamHI) and 3' (SacI), was cloned into the vector pESC-URA (Ampicillin) by 5' BamHI and 3'SacI (using recombination). The plasmid pACT1p-scFv-GFP-actin-A D H A t (URA), and one copy of mini-scale recombinant plasmid DNA was prepared and divided into 1 tube and 1 tube enacentesis containing the recombinant plasmid.

![](https://static.igem.wiki/teams/4203/wiki/experiment11.jpg)
Optimized pACT1p plasmid

Testing stage

[See Project-Experimence](https://2022.igem.wiki/bjwz-china/experiments)

Learning stage

We successfully constructed pTDH3p, pADH1, and pACT1p plasmids during the experimental stage, and verified the correct construction of pACT1p and pTDH3p plasmid, while allowing them to successfully express the fusion protein in BY4741. However, for objective reasons, the pADH1 plasmid construction period was long, leading to some delay in our experimental planning. At the same time, BY4741 is more fragile and its growth cycle is longer. Our time is not enough for us to successfully transfer the three plasmids into BY4741 together to verify the effect of sound on it. So the second best thing is to transfer the single plasmid into BY4741. The successful expression of the two existing plasmids makes us believe that our vision is achievable.

Documentary references

[1]A Protein-Tagging System for SignalAmplification in Gene Expression and Fluorescence Imaging
Marvin E. Tanenbaum, Luke A. Gilbert,Lei S. Qi, Jonathan S. Weissman, and Ronald D. Vale1
[2]Yu Hong, Guangqing Lu, Jinzhi Duan, Wenjing Liu and Yu Zhang.Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imaging
[3]Yung-Hsin Huang1, Jianzhong Su, Yong Lei, Lorenzo Brunetti, Michael C. Gundry, Xiaotian Zhang,Mira Jeong, Wei Li and Margaret A.Goodell1.DNA epigenome editing using CRISPR-CasSunTag-directed DNMT3A
[4]Xiaoying Chen, Jennica L. Zaro, Wei-Chiang Shen .Fusion protein linkers: Property, design and functionality
[5]Sumiyo Morita, Hirofumi Noguchi, Takuro Horii1, Kazuhiko Nakabayashi, Mika Kimura1
Kohji Okamura, Atsuhiko Sakai, Hideyuki Nakashima, Kenichiro Hata, Kinichi Nakashima & Izuho Hatada.Argeted DNA demethylation in vivo using dCas9-peptiderepeat and scFv-TETTETTET1 catalytic domain fusions
[6]Qi Jieqiong, Gao Hong, Zhuang Tingting, Yu Wengong, Gu Yuchao. Recombinant expression and activity detection of the single-chain antibody scFv-GCN4 in Escherichia coli [R]. Shandong: School of Medicine, Ocean University of China, Qingdao, Shandong province266000
[7]YAO Longfang,ZHOU Li,WANG Jie,CAI Liang.Visualize Cytoplasmic Abundant ProteinLocalization via SunTag Strategy
( Collaborative Innovation Center of Genetics and Development,State Key Laboratory of Genetic Engineering,School of Life Sciences,Fudan University,Shanghai 200438,China)
[8]Xiaoshu Xu1 and Lei S. Qi1,A CRISPR-dCas Toolbox for GeneticEngineering and Synthetic Biology
[9]A TRPV family ion channel requiredfor hearing in Drosophila
Janghwan Kim, Yun Doo Chung, Dae-young Park, SooKyung Choi,Dong Wook Shin, Heun Sohk, Hye Won Lee, Wonseok Son,Jeongbin Yim{, Chul-Seung Parkk, Maurice J. Kernan? Changsoo Kim
[10]Benot Kornmann1, Erin Currie1, Sean R. Collins, Maya Schuldiner, Jodi Nunnari,Jonathan S. Weissman, and Peter Walter.An ER-Mitochondria Tethering Complex Revealed by a Synthetic Biology Screen
[11]An Introduction to the Genetics andMolecular Biology of Yeast
[12]development and application of CRISPR-based gene activation 张鑫
[13]Distinct Roles of TRP Channels in AuditoryTransduction and Amplification in Drosophila Brendan P. Lehnert, Allison E. Baker, Quentin Gaudry, Ann-Shyn Chiang, and Rachel I. Wilson
[14]Nanchung and Inactive define pore properties of the native auditory transduction channel in Drosophila
Bingxue Li, , Songling Li , Honglan Zheng and Zhiqiang Yana
[15]Prestin is an anion transporter dispensable for mechanical amplification in Drosophila hearing
Ryan G. Kavlie · Janice L. Fritz · Florian Nies · Martin C. G?pfert · Dominik Oliver · Joerg T. Albert · Daniel F. Eberl
[16]Sound response mediated by the TRP channels NOMPC,NANCHUNG, and INACTIVE in chordotonal organs of Drosophila larvae
Wei Zhang, Zhiqiang Yan, Lily Yeh Jan, and Yuh Nung Jan1
[17]T cell stimulation and expansion by SunTag-based clustering of anti-CD3/CD28 scFv
Kunhong Zhong1,\*, Zhiyong Liu2,\*, Hongjian Li1, Shasha Zhao1, Yuelong Wang2, Wenhao Guo1, Xi Zheng, Hui Yang, Gang Guo, Liangxue Zhou, Jianguo Xu, Aiping Tong
[18]Two Interdependent TRPV Channel Subunits, Inactive and Nanchung, Mediate Hearing in Drosophila Zhefeng Gong,1\*Wonseok Son, Yun Doo Chung, Janghwan Kim, Dong Wook Shin, Colleen A. McClung,Yong Lee, HyeWon Lee,Deok-Jin Chang, Bong-Kiun Kaang, Hawon Cho, Uhtaek Oh,Jay Hirsh,Maurice J. Kernan, and Changsoo Kim
[19]A CRISPR-dCas Toolbox for Genetic Engineering and Synthetic Biology Xiaoshu Xu1 and Lei S. Qi
[20]Yang Jiwang, Li Lin, He Chunlan, Pan Yanting, Zhang Gangping, Zhou Ping, Huang Yaoxiong. Fluorescence image analysis of S. cerevisiae in S. cerevisiae. [R]. Guangdong: Department of Biomedical Engineering, School of Life Science and Technology, Jinan University

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![](https://static.igem.wiki/teams/4203/wiki/experiment.jpg)

【1】Experimental preparation

[1]Amplify the plasmid of interest
Two of the synthesized expression plasmids (pACT1 and pTDH3) were transformed into DH5α, and the strains containing the target plasmids were screened out. The plasmids were extracted after 18 hours of expansion at 37℃ and 180rpm.

[2]Extracted the target plasmid

\* Add 0.2~0.5ml Buffer P1 to RNase A dry powder, beat and mix for 5~10 times to fully dissolve the RNase A, and then all the RNase A is transferred to Buffer P1 and stored at 2-8℃.

\* Buffer PW2 was stored at room temperature by adding 4 x volume of absolute ethanol as indicated by the bottle label.

\* Sterilized 1.5-m l centrifuge tube and used for DNA collection.

\* LB medium and the corresponding culture tubes or culture flasks.

\* Below centrifugation was performed at room temperature and column blockage at low temperatures.

1.The plasmid-containing strains were seeded in 50ml tubes containing 10 m l of L A medium and incubated in a 37℃ shaker for 12 to 16 h to amplify the plasmid.

2.The ls were centrifuged at 3,000~5,000 g for 10 min and 10 to 15 m l of broth were collected.

3. Pour down the medium and buckle the absorbent paper and gently beat the residual liquid. Add 500 μl Buffer P1/RNase A mixed liquid and resuspended. Before use, ensure that the RNase A is added to the Buffer P1. Thorough resuspension of bacteria is quite important to achieve high yield, and cell clumps should be invisible after resuspension.

4. The resuspension was transferred to a 2-m l centrifuge tube. Add to the 500 μl Buffer P2. Gently upside down 8 to 10 times. Place at room temperature for 2 minutes, occasionally reverse several minutes. Gently reverse the mixing, do not vortex, otherwise it will cause genomic DNA breakage and contamination. When the amount of bacterial solution exceeds 10ml, the lysate will be very viscous and difficult to mix. After full lysis, the solution becomes viscous and bright. If necessary, slowly reverse the lysate until the lysate becomes clear, but not over 4 minutes.

5. Add 700 μl Buffer P3 and immediately reverse and mix well 15~20 times. After adding Buffer P3, the mixing should be reversed immediately to prevent precipitation agglomeration and affecting the neutralization effect. The mixing process must be gentle. When the amount of bacterial liquid is more, neutralization will be more difficult to proceed, increase the number of inverted to the solution thoroughly neutralization.

6.It was centrifuged at 6. 13,000 ×g for 10 min.

7. Cover the HiPure DNA Mini Column III column in the collection tube. Transfer half the volume of the supernatant to the column. The mixture was centrifuged at 13,000 g for 30~60 seconds.

8. Pour away the effluent and put the column back into the collection tube. Transfer the remaining supernatant to the column. The mixture was centrifuged at 13,000×g for 30~60 seconds.

9. Pour away the effluent and put the column back into the collection tube. Add the 500 μl Buffer PW1 to the post. The mixture was centrifuged at 13,000×g for 30~60 seconds. This step can be omitted in treating nuclease knockout strains (end A-), such as JM109 et al. Treatment with nuclease-rich strains (end A +) such as HB101 cannot be omitted. The Buffer PW1 contains protein denaturants, please wear gloves to treat it. HiPure Plasmid Plus Kits is recommended when treating nuclease-containing strains to improve plasmid stability.

10. Pour the effluent, put the column back into the collection tube and add 600 μl Buffer PW2 to the column.13,000 ×g centrifugation for 30-60 seconds.

11. Pour the effluent, put the column back into the collection tube and add 600 μl Buffer PW2 to the column.13,000 ×g centrifugation for 30~60 seconds.

12. Pour the filtrate and put the column back into the collection tube. The drying column was centrifuged at 13,000 ×g for 2 min. Don't ignore this step. This step was taken to remove the residual ethanol in the column.

13. Cover the column in a sterilized 1.5ml centrifuge tube. Add 75~100 μl Elution Buffer or sterilized water to the center of the membrane of the column. Let for 2 min and centrifuged at 12,000 ×g for 1 min.

14. Leave the column and save the plasmid at-20℃.

[3]Quantitative plasmids were detected by electrophoresis
1. Take 2.5 μl Loading Buffer, mix 1 μl plasmid and drop into the sample hole.

2.10,000 bp Marker was added for control.

3. Agarose gel was put into electrophoresis and run at 180v for 25min.

4. Plasmid was extracted using automatic gel image system.The target plasmid was verified by enzyme digestion

 [4]The target plasmid was verified by enzyme digestion

 1) pACT1p enzyme digestion and verification:

Xho I: 9149 bp

Hind Ⅲ: 2991 bp+6158 bp

Sal I :3031 bp+6118 bp
2) pTDH3p enzyme digestion and verification:

 Xho I: 12534 bp

EcoR I :3970 bp+8564 bp

EcoR V: 7367 bp+5167 bp

\*System configuration:

The dna sample was used in 10 μl

Cutting buffer was prepared at 1.5 μl

Enzyme:1 μl

Sterile water was in 2.5 μl

1.Plasmid samples were taken in a 0.5-m l centrifuge tube

2. Mix well; centrifuge at 4000rpm for 30s and swing to the bottom of the tube

3.37℃, and digested for 2h

4.65℃, insulated for 20min

5. Electrophoresis verification: take 1μl amplification product, mix with 2.5 μl 10 × loading buffer, add to the point sample hole with a pipette, electrophoresis at 180v for 25min, and remove the gel. In control with Marker, the band size was determined with the correct transformants

 [5]Medium preparation:

 SD medium (200 ml)

YNB:10 ml

Glucose: 4 g

Agar: 4 g

Methionine: 0.004 g

Histidine: 0.004 g

Leucine: 0.012 g

Uracil: 0.008 g

dH2O: 200 ml

1. Glucose, add 4 g of agar each to a tapered flask, 185m l of distilled water and autoclaved at 115 ℃ for 15 min

2. Ultra-clean table disinfection and ultraviolet test extinguishing tube (set 10ml scale line with water in advance)

3. Corresponding corresponding amino acids and uracil, loaded into 5 ml tube and added 5ml water to dissolve.

4. Filter the amino acids and uracil with a sterile filter membrane and connect them to a sterile tube

5. Take 10 ml of YNB from the ultra-clean table to the test tube, add YNB solution and amino acid uracil solution to the autoclaved tapered bottle, and shake at 50 ℃ for half an hour to dissolve

【2】Yeast transformation

![](https://static.igem.wiki/teams/4203/wiki/experiment12.jpg)
[1]Single plasmid yeast transformation

(1)Take 100μl For the BY4741 competent cells melted on the ice, pre cooled target plasmid 2g and Carrier DNA 10 μl, PEG / LiAc 500μl .Suck and beat for several times, mix well, and take a 30℃ water bath for 30 min (turn over for 6-8 times when 15 min)
Note: Carrier DNA 96 ℃ water bath for 3 min, fast ice bath for 3 min, repeat once
(2) Move the competent state to 42 ℃ water bath for 15 min (turn it over for 6-8 times and mix well at 7.5 min).
(3) Centrifuge the supernatant at 5000 rpm for 40s, ddH2O 400μl Suspend again, centrifuge for 30s and discard the supernatant.
(4) ddH2O 50μl Heavy suspension, coated plate, culture at 29 ℃ for 48-96 h.

[2]Yeast transformant colony PCR and electrophoresis verification

 • two × Fast PCR Mix 10 ul

• Primer F:1 ul

• Primer R:1 ul

• Bacterial liquid: 2 ul

• ddH2O:6 ul

(1) Add 6μl ddH2O to the PCR tube , take the convertor with uniform size from the plate and add it.

(2) Take 2μl The bacterial solution was cultured in defective SD medium at 29℃ for 180rpm.

(3) Take 4μl for PCR validation, 2μl Bacterial liquid test and the rest for standby.

(4) PCR amplification was carried out according to the system.

(5) Take 1μl Expansion products and 2.5μl10 \* loading buffer, mix well, add it into the sample hole with a pipette, conduct 180v electrophoresis for 25min, and take out the gel.

(6) Compare with Marker to determine the stripe size and correct transformant.

[3]Determine the growth curve of single plasmid yeast, and verify that single plasmid has no significant effect on yeast growth

(1) Take single colonies of BY4741, pTDH3p and pACT1p into the liquid SD medium, and conduct amplification and culture at 29 ℃ at 180rpm until the OD660 value is 0.2.

(2) Take 2% as the inoculum quantity to connect the bacteria to the liquid SD culture medium that has been repackaged. One kind of bacteria is connected to three bottles of culture medium to take the average value of multiple experiments, and then expand and culture at 29℃ at 180rpm.

(3) The OD660 value shall be measured by 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.

(4) Summarize the data and draw a curve.

[4]Fluorescence observation of single plasmid yeast

Fluorescence observation of transformants: 100μl was taken from the 5ml EP tube shaken for 3~4 days, washed with 1ml PBS, 4000rpm, 1min, discarded supernatant, resuspended with a new 50~100μl PBS, and 10μl of the resuspended transformants solution was prepared for observation. Then, turn on the microscope and software ZEN3. 4. The corresponding GFP and DAPI channel were used for fluorescence observation.

[5]Glycerol tube strain preservation
1、Preservation of BY4741 and BY4741-DmIav, BY4741-ACT1

(1)50ml of bacterial solution was separately packed into 2ml finger tubes, centrifuged at 5000g for 5-10min, and the supernatant was discarded.

(2) Add 500ml of fresh SD culture medium into each finger tube, resuspension and merge into one tube.

(3) After centrifugation, discard the supernatant and add 1ml fresh SD medium to resuspension.

(4) Under aseptic operation conditions, add 800mL of bacterial solution and 1200mL of 50% glycerin solution into the sterilized 2mL cryopreservation tube, mix gently and fully;

(5) Store in - 80 ℃ refrigerator.

2、Preservation of pTDH3p and pACT1p plasmids

(1) 50ml of bacterial solution was separately packed into 2ml finger tubes, centrifuged at 5000g for 5-10min, and the supernatant was discarded.

(2) Add 500ml fresh LB culture medium into each finger tube, resuspension the cell and merge it into one tube.

(3) After centrifugation, discard the supernatant and add 1ml fresh LB medium to resuspension.

(4) Under aseptic operation conditions, add 1000mL bacterial solution and 1000mL 50% glycerol solution into the sterilized 2mL cryopreservation tube respectively, mix gently and fully;

(5) Store in - 80 ℃ refrigerator.

[6] Oil mirror making
For 30 ml of BY4741-ACT1 transformants, BY4741-DmIav transformants and BY4741 cultured at 29 °C, 180 rpm for 72 h, we made temporary mounts for observation.
1. Wipe the slides. Mix 200 μl of the bacterial solution with 200 μl of ink and drop it onto the slide with a dropper.
2. After the low magnification field of view is adjusted accurately, the temporary film is adjusted from the low magnification mirror to the 100 times oil mirror.
3. Turn the rough adjuster to make the oil lens drop and immerse in the glycerol, gently contact the slide.
4. Adjust the lens to make the field of view clear.
5. Move the thruster and turn the micro-adjuster to observe the specimen. Look for areas with dense yeast and take pictures with your phone.
6. After observation, turn the rough adjuster to raise the lens barrel and remove the slide. Immediately wipe the glycerin off the lens with a mirror polishing paper.

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![](https://static.igem.wiki/teams/4203/wiki/results-post.jpg)

【1】experimental result

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.

![](https://static.igem.wiki/teams/4203/wiki/result-new.jpg)

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.

 ![](https://static.igem.wiki/teams/4203/wiki/result-new1.jpg)

 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.

![](https://static.igem.wiki/teams/4203/wiki/result-3.png)

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

![]( https://static.igem.wiki/teams/4203/wiki/results4.png)

(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.

![](https://static.igem.wiki/teams/4203/wiki/results5.png)

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.

![](https://static.igem.wiki/teams/4203/wiki/resultsa.png)

Figure 6 （BY4741-ACT1，72h）

![](https://static.igem.wiki/teams/4203/wiki/resultsnew.png)

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

![](https://static.igem.wiki/teams/4203/wiki/resultsb.png)

Figure 8（ BY4741，72h）

![](https://static.igem.wiki/teams/4203/wiki/resultsnew1.png)

 Figure 9 (BY4741 100x oil microscopic examination)

![](https://static.igem.wiki/teams/4203/wiki/resultsnew2.png)

Figure 10（BY4741-DmIav 72h）

![](https://static.igem.wiki/teams/4203/wiki/resultsnew3.png)

Figure 11 (BY4741-DmIav oil mirror observation)

![](https://static.igem.wiki/teams/4203/wiki/line.jpg)

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

![](https://static.igem.wiki/teams/4203/wiki/result-13.jpg)

 Figure 13 Mingchang

![](https://static.igem.wiki/teams/4203/wiki/result-new2.jpg)

 Figure 14 Fluorescence

![](https://static.igem.wiki/teams/4203/wiki/result-15.jpg)

 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

![](https://static.igem.wiki/teams/4203/wiki/result-16.jpg)

Figure 16 Bright field

![](https://static.igem.wiki/teams/4203/wiki/result-17.jpg)

)Figure 17 Fluorescence

![](https://static.igem.wiki/teams/4203/wiki/result-18.jpg)

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.

【2】 Future experiment plan

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.

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