Silver Medal Experiment:
I. Design and construction of expression plasmids pRS415-CMPG/CMPG Pro, pRS415-CMPS/CMPS Pro
1. pCUP1 , mfα2, CYC1, pprm1, Ste5ΔN-CTM, pprm1 pro fragments were synthesized by the company.
2. Hind III digested plasmid pRS415- CMPG/CMPG Pro, Not Ⅰ digested plasmid pRS415-CMPS/CMPS Pro
3. Validated by agarose gel electrophoresis
4. Homologous recombination of fragments and vectors into E. coli DH5α and transfection of fragments into E. coli DH5α
5. Chemical transfer of recombinant products into E. coli DH5α
II. Construction of recombinant strains BY4741-pRS415-CMPG/CMPG Pro, pRS415-CMPS/CMPS Pro
Prepare receptor cells of Saccharomyces cerevisiae BY4741, and introduce the recombinant plasmid into the yeast cells by electrotransfer. Gradient diluted, and coated on SC(-Leu) plates.
III. Validation of recombinant plasmids pRS415-CMPG/CMPG Pro, pRS415-CMPS/CMPS Pro
Single colonies were picked from the chemotaxis plates of E. coli for culturing access to test tubes containing 5mL LB+5μL of Amp medium, incubated in a shaker for 12-16h. Then the plasmids were extracted to be used as templates for plasmid PCR. After verification by agar gel electrophoresis, the correct samples were sent for sequencing.
IV. Validation of recombinant strains BY4741-pRS415-CMPG/CMPG Pro, pRS415-CMPS/CMPS
Pro A number of single colonies on yeast electrotransfer plates were selected for transpointing, followed by colony PCR, with primers using M13, colony PCR, and finally validation by agarose gel electrophoresis.
V. Copper ion induced GFP expression and quantitative analysis
1. Strain pre-culture In an ultra-clean table, take the bacterial solution, scribe lines on a SC(-Leu) plate and label it. The delineated plates were incubated in a constant temperature incubator at 30°C for 1-2 days for strain recovery. Pick single colonies of recombinant yeast strains BY4741-pRS415-CMPG/CMPG Pro, BY4741-pRS415-CMPS/CMPS Pro on the plates and inoculate into 5 mL of SC(-Leu) liquid medium and label it. Incubate the inoculated liquid medium in a constant temperature shaker for about 18h.
2. Copper ion induced expression of GFP and flow cytometry analysis Determine the OD600 value of the bacterial solution by UV spectrophotometer, and add a certain amount of bacterial solution into 2mL SC(-Leu) liquid medium so that the OD600 value become 0.1. Then dilute 25mM CuSO4 mother liquor according to the ten-fold gradient and add different concentrations of copper ions into the 2mL bacterial solution with the OD600 value of 0.1. The inoculated test tubes were put into a constant temperature shaker for 24h, and the bacterial solution after 24h of copper ion induction was ready for sample processing and detection by flow cytometry.
3. Time expression spectrum of sensor response to copper ions Use UV spectrophotometer to measure the OD600 value of the pre-cultured bacterial solution, add 100mL SC(-Leu) medium to the sterilized 250mL conical flask, and the bacterial solution was added so that the starting OD600 value of the final culture fluid system was 0.1. Then a certain amount of 25mM CuSO4 was added to make the final concentration of copper ions in the culture solution was 10 μM. The conical flask was placed into a constant-temperature shaking bed to cultivate the bacterial solution. After that, samples were taken every 2h, OD600 was measured by UV spectrophotometer and GFP expression was measured by enzyme marker until 24h. Sample processing was performed and the sample was detected using flow cytometry.
4. Specificity of the copper ion sensor Prepare ZnSO4 , MgSO4 , MnSO4 solution of25 mM respectively, and determine the OD600 value of the pre-culture bacterial solution by UV spectrophotometer. Add a certain amount of bacterial solution to the 2mL SC(-Leu) liquid medium, so that the OD600 value of the final culture system was 0.1. Then dilute CuSO4 , ZnSO4 , MgSO4 , MnSO4 solution of 25 mM according to the Ten-fold gradient dilution, add a certain amount of CuSO4 , ZnSO4 , MgSO4 , MnSO4 solution to the 2mL bacterial solution with OD600 value of 0.1 , so that the metal ions concentration of the final culture system was 10 μM. The inoculated tubes were placed into a constant temperature shaker incubation for 24h to process the samples, and then subjected to flow cytometry.
Gold Medal Experiment:
I. Machine learning-assisted assembly of inducible and constitutive UAS elements
Structural analysis of the pprm1 promoter by running the NuPoP algorithm (https://bioconductor.org/packages/NuPoP/) revealed 2 features in the pprm1 promoter: first, it contains multiple nucleosome affinity reduction sequences, which help to enhance promoter activity; The second is that it contains three short consensus PREs. We defined pprm1 from -280bp to -101bp upstream of the gene start codon as UASprm1, and UAScit at 275nt, as the upstream activation sequence of citrate synthase-encoding gene cit1, which is one of the transcriptional enhancers that have been identified in Saccharomyces cerevisiae. Place these two UAS elements immediately upstream of the original pprm1 to form a new promoter, UASprm1-prm1 and UAScit-prm1 were constructed to obtain high-intensity promoter variants.
II. Design and construction of expression plasmid pRS415-UASprm1-prm1-GFP-CYC1/pRS415-UAScit-prm1-GFP-CYC1
1.UASprm1-prm1/UAScit-prm1 promoter fragment was synthesized through the company.
2. Plasmid pRS415-GFP-CYC1 was digested by BamHⅠ.
3. Verified by agarose gel electrophoresis
4. Homologous recombination of fragments and vectors.
5. Transfect the recombinant product into E. coli DH5α.
III. Validation of recombinant plasmid pRS415-UASprm1-prm1-GFP-CYC1/pRS415-UAScit-prm1-GFP-CYC1
Single colonies were picked from the chemotaxis plates of E. coli for culturing access to test tubes containing 5mL LB (Amp) medium and incubated in a shaker for 12-16h. Then the plasmids were extracted and used as templates for plasmid PCR. After verification by agar gel electrophoresis, the correct samples were sent for sequencing.
IV. Validation of recombinant strain pRS415-UASprm1-prm1-GFP-CYC1/pRS415-UAScit-prm1-GFP-CYC1
Dilute the yeast and prepare receptor cells of Saccharomyces cerevisiae BY4741. Plasmids were electro-transferred into yeast cells. After gradient diluted, the cells were coated on SC(-Leu) plates. A number of single colonies on the yeast electro-transfer plates were selected for trans-spotting, followed by colony PCR, and finally verified by agarose gel electrophoresis. The samples were sent for testing and the correct strains were preserved.
V. Fluorescence assay of recombinant strain
pRS415-UASprm1-prm1-GFP-CYC1/pRS415-UAScit-prm1-GFP-CYC1 A single colony was picked and connected to SC (-leu) liquid medium, cultured for 20 h. Then dilute it to OD600 of 0.1, cultured at 30℃ for 3 h. Add 5µM pheromone, induced for 1 h, and washed three times by 1×PBS. Detect by flow cytometry.