Plasmid design

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.
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.
pTDH3p plasmid

(1)SunTag
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.
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

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.

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.

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.

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.

Optimized pACT1p plasmid

See Project-Experimence

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.

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[17]T cell stimulation and expansion by SunTag-based clustering of anti-CD3/CD28 scFv
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