June
June 1st
Protein Engineering Module
Purification of TFD and TFD-S protein
SDS-PAGE polyacrylamide gel electrophoresis verification of TFD and TFD-S
June 7th
Protein Engineering Module
Test of lanthanide ion adsorption capacity of TFD and TFD-S
July
July 14th
Preparation of experimental materials: preparing LB and YPD medium
Yeast Surface Display Module
Preparation of single colonies of E. coli containing the pYD1-TFD-SS plasmid
July 15th
Protein Engineering Module
Inoculation of E. coli containing pET-28a (+) -TFD-S plasmid
Design and synthesis of primers for targeted mutagenesis of TFD sequences on pET-28a (+) plasmid（I6E-F/R、I52E-F/R、I175E-F/R、I221E-F/R）
Yeast Surface Display Module
Inoculation of E. coli containing pYD1-TFD-SS plasmid
July 16th
Preparation of experimental equipment:
SD medium (Saccharomyces cerevisiae minimal medium, glucose only)
Yeast-selective nutrient-deficient media
Protein Engineering Module
Extraction and sequencing of the plasmid pET-28a (+)-TFD-S (primers T7-seq-F/ R)
Mutate I6E, I52E, I175E, and I221E of the TFD sequence on the pET-28a (+)-TFD-S plasmid separately
Yeast Surface Display Module
Extraction and sequencing of the plasmid containing pYD1-TFD-SS (primer pYD1-seq-F/R)
July 17th
Protein Engineering Module
Pick and sequence single E. coli colonies with single mutations of I6E, I52E, I175E, and I221E, colony PCR
Yeast Surface Display Module
Preparation of reagents required for yeast transformation
Amplified chassis strain for surface display: Saccharomyces cerevisiae EBY100
Membrane Bio-Reactor & Biosafety Module
Extraction of genomic DNA from Saccharomyces cerevisiae BY4741
Extraction and sequencing of Saccharomyces cerevisiae integration plasmid pUMRI-pTEF1
July 18th
Protein Engineering Module
The successful gene mutations are: 6-1, 6-2, 6-3, 52-2, 52-3, 175-1, 175-3, 221-1, 221-2, for a total of 9
Yeast Surface Display Module
Preparation of SC-TRP solid medium
Transformation of Saccharomyces cerevisiae EBY100 by lithium acetate with pYD1 plasmid and pYD1-TFD plasmid respectively
Membrane Bio-Reactor Module
Design of PCR primers for FLO11 gene
Lysis buffer directly lyses yeast cells to extract yeast DNA
Biosafety Module
Amplification of the Saccharomyces cerevisiae CEN.PK2–1C strain containing the pCTR3 promoter
July 19th
Membrane Bio-Reactor Module
Construction of recombinant plasmids pUMRI-10-pTEF1-FLO11-tCYC1 for gene integration by restriction digestion and enzyme ligation
July 21st
Configuration of LB (Amp), LB (Kan) solid medium
Yeast Surface Display Module
Preparation of SC-TRP liquid medium
Membrane Bio-Reactor Module
Extraction of plasmid pUMRI-10
Biosafety Module
Extraction of plasmid p416-pTEF1-Cas9-tCYC1-G418
Construction of recombinant plasmid p416-pCTR3-mCherry-tCYC1 by Gibson assembly method
July 22nd
Protein Engineering Module
Testing TFD, TFD-S tryptophan-enhanced Tb luminescence with Ce, Eu, Yb substitution.
Biosafety Module
Colony PCR verified the plasmid construction results of p416-pCTR3-mCherry-tCYC1
July 23rd
Protein Engineering Module
Reset the concentration gradient, test TFD and TFD-S, use tryptophan to enhance the luminescence of Tb, and replace Ce, Eu, and Yb.
Yeast Surface Display Module
Determine the OD600 value of yeast (pYD1-TFD and pYD1) grown in SC-TRP liquid medium; centrifuge, wash, resuspend the cells to an OD600 value of about 0.75, and shake the cells.
Biosafety Module
Extraction and sequencing of plasmid p416-pCTR3-mCherry-tCYC1 1~4
July 24th
Membrane Bio-Reactor Module
Preparation of complete YPD medium supplemented with G418
Preparation of pTEF1 Donor
Construction of p426-SpSgh and gRNA1-4 restriction ligation product; failed
Biosafety Module
The plasmid containing the pCTR3-mCherry-tCYC1 expression cassette constructed by Gibson is successfully constructed and transformed into Saccharomyces cerevisiae EBY100.
July 25th
Yeast Surface Display Module
Take the culture medium of EBY100 (pYD1) and EBY100 (pYD1-TFD) after 48h of culture, measure the OD600 value, and continue to culture for 60 hours.
Membrane Bio-Reactor Module
Verify that CRISPR has integrated into the correct sequence at the pTEF1 location.
Sequencing verification of unpurified PCR products of FLO11-Promoter
July 26th
Yeast Surface Display Module
Measure Tb(Ⅲ) binding in EBY100 (pYD1) and EBY100 (pYD1-TFD) strains
Membrane Bio-Reactor Module
Re-extraction of p426-SpSgh plasmid
Construction of p426-SpSgh and gRNA1-4 restriction endonuclease ligation products
July 27th
Yeast Surface Display Module
Re-induced expression of surface display protein, TFD
Membrane Bio-Reactor Module
Cultivation and sequencing of p426-SpSgh BsaI digestion products
Biosafety Module
Re-transformation of single colonies using Saccharomyces cerevisiae BY4741
July 28th
Yeast Surface Display Module
Proliferation and culture of EBY100, EBY100 (pYD1), and EBY100 (pYD1-TFD)
Membrane Bio-Reactor Module
The gRNA plasmids of 1-1, 1-2, 1-3, 1-4, 2-2, 2-4, 2-5, 3-1, 3-2, 3-3, 3-5, 4-3, and 4-4 are successfully constructed.
Obtain the full-length FLO11 gene from the genome of Saccharomyces cerevisiae BY4741 by PCR
July 29th
Membrane Bio-Reactor Module
The pUMRI-10-pTEF1 plasmid and the FLO11 gene PCR purified product are double-digested with BamHI and KpnI.
Biosafety Module
Amplification of Saccharomyces cerevisiae BY4741 transformed with p416-pCTR3-mCherry-tCYC1
July 30th
Membrane Bio-Reactor Module
Connect the FLO11 gene to the vector plasmid pUMRI-10-pTEF1 and transform it into E. coli
Use CRISPR/Cas9 technology to integrate the pTEF1 promoter upstream of the FLO11 gene.
Transform Saccharomyces cerevisiae BY4741 using the lithium acetate method, and simultaneously introduce Cas9 expression plasmid, gRNA expression plasmid, pTEF1 Donor, and coat SC-URA+G418 plates.
Biosafety Module
Designing an inhibitory concentration gradient to test the pCTR3 promoter
July 31st
Bioleaching Module
Construction and sequencing of single colonies of the pUMRI-10-pTEF1-FLO11-tCYC1 plasmid
Yeast Surface Display Module
Re-expression of proteins
Biosafety Module
Saccharomyces cerevisiae BY4741 with p426-pCTR3-mCherry-tCYC1 free plasmid was cultured in shake flasks with the concentration gradient of 0, 0.2, 0.5, 1, 2, 10, 20, 30, 40, 50, 60 μM of copper ion, and control group was set up. Fluorescent detection is performed every 12 hours.
August
August 1st
Protein Engineering Module
Transformation of pET-28a (+)-TFD-M with four mutations plasmid into E. coli
Yeast Surface Display Module
Expanded culture of EBY100, EBY100 (pYD1), EBY100 (pYD1-TFD)
Membrane Bio-Reactor Module
Extraction and sequencing plasmid pUMRI-pTEF1-FLO11-tCYC1 1/3/5
Preparation of SC+FOA medium
August 2nd
Protein Engineering Module
Single colony amplification of E. coli transformed with pET-28a (+)-TFD-M
Membrane Bio-Reactor Module
Colony PCR confirms that CRISPR gene integration failed
Preparation of Donor DNA for CRISPR Gene Integration
August 3rd
Protein Engineering Module
Extraction of pET-28a (+)-TFD-M with four mutations plasmid
Mutation of Q154W and Q323W in pET-28a (+)-TFD-M plasmid by circular PCR
Membrane Bio-Reactor Module
Gibson assembly to construct plasmid pUMRI-pTEF1-FLO11-tCYC1 for gene integration
August 4th
Protein Engineering Module
The mutant plasmids Q154W-2, Q154W-3, Q323W-3, Q154W-Q323W-1, and Q154W-Q323W-4 are successfully constructed
Yeast Surface Display Module
The expression of EBY100, EBY100 (pYD1), and EBY100 (pYD1-TFD) proteins is induced for 60 hours, and the culture medium is collected.
August 6th
Protein Engineering Module
Transformation of the plasmid with two mutations Q154W-Q323W into E. coli
August 7th
Protein Engineering Module
E. coli single colony transformed with two mutations Q154W-Q323W plasmid are cultured for 12-16h in test tubes.
August 8th
Protein Engineering Module
Inoculate 1% bacterial solution into a shake flask and culture until OD600 is 0.6-0.8, then add protein expression inducer for further culture.
Membrane Bio-Reactor Module
Extract yeast genome, extract FLO11 gene, failed
Concentrate the system containing the FLO11 gene by ethanol precipitation, failed
pTEF1 Donor sent for sequencing, failed
August 9th
Protein Engineering Module
Protein purification and electrophoresis verification
Membrane Bio-Reactor Module
Extract the yeast genome, extract the FLO11 gene, and then concentrate.
August 10th
Membrane Bio-Reactor Module
Plasmid transformation into E. coli
August 11th
Protein Engineering Module
Measurement of TFD-QQ, TFD-S, and TFD-M protein concentrations
Membrane Bio-Reactor Module
Colony PCR verification of E.coli, Gibson assembly result seems to be correct.
August 12th
Yeast Surface Display Module
Re-run flow cytometry
Membrane Bio-Reactor Module
Sequencing of Gibson assembly products
August 13th
Protein Engineering Module
Protein characterization experiments using the microplate reader were unsuccessful due to the time limit.
Membrane Bio-Reactor Module
Sequencing results are wrong. Re-PCR of FLO11 showed no bands.
August 14th
Protein Engineering Module
The fluorescence of TFD protein bound to terbium ion was measured by the microplate reader.
Yeast Surface Display Module
Yeast cells were re-cultured for protein electrophoresis verification
Membrane Bio-Reactor Module
Re-PCR of the full-length FLO11 gene
August 15th
Protein Engineering Module
The fluorescence of TFD protein bound to terbium ion was measured by the microplate reader.
August 16th
Protein Engineering Module
The four-mutant TFD-M plasmid is subjected to gene mutation to mutate the original binding site back to the TFD-QQ state.
August 17th
Protein Engineering Module
Site-directed mutagenesis
The pET-28a (+)-TFD-M plasmid with six mutations is subjected to gene mutation to mutate the original binding sites back to the TFD-QQ state; Get two plates: E35V-E204V and E158K-E327K.
August 18th
Protein Engineering Module
Pick single colonies to get E35V-E204V TFD plasmid and E158K-E327K TFD plasmid, and send them for sequencing.
August 19th
Membrane Bio-Reactor Module
Donor DNA for CRISPR/Cas9 gene integration of the pTEF1 promoter is prepared by PCR, and the electrophoretic bands are correct, ethanol precipitation is concentrated.
August 20th
Membrane Bio-Reactor Module
Integration of pTEF1 promoter by CRISPR/Cas9
August 22nd
Yeast Surface Display Module
Surface displayed yeast strains: ① DTT treatment to break disulfide bonds, ultrafiltration, SDS-PAGE protein gel electrophoresis verification; ② After yeast cell rupture, protein column purification, ultrafiltration concentration, SDS-PAGE protein gel
August 23rd
Yeast Surface Display Module
Obtain gene and vector fragments for Gibson assembly by PCR.
Membrane Bio-Reactor Module
Single colonies grow on plates with the CRISPR/Cas9 gene integrated with the pTEF1 promoter, and colony PCR is performed on plates containing gRNA1-4 and verified by electrophoresis.
August 24th
Yeast Surface Display Module
Connect the Aga2 and Aga2-TFD fragments on the pYD1 plasmid to the pESC-TRP high-copy plasmid vector, the 6×His-Agα1-GS linker-TFD fragment to the panARS-HZP plasmid vector by Gibson assembly; transform them into E. coli
Membrane Bio-Reactor Module
Removal of CRISPR/Cas9 selection marker
Connect the FLO11 gene to the vector pUMRI-10 by Gibson assembly; transform it into E. coli.
August 25th
Yeast Surface Display Module
Verify the construction results of plasmid pESC-TRP-Aga2、pESC-TRP-Aga2-TFD and panARS-HZP-6×His-Agα1-GS linker-TFD by colony PCR, and verify the PCR products by electrophoresis.
Membrane Bio-Reactor Module
Verify the construction results of plasmid pUMRI-10-pTEF1-FLO11-tCYC1 by colony PCR, and verify the PCR products by electrophoresis.
August 26th
Yeast Surface Display Module
The two fragments are assembled using Gibson, and the plasmid vector is obtained by PCR using the panARS-HZP-6×His-TFD- GS inker-Agα1 plasmid as a template (failed), and the α-signal peptide fragment is obtained by PCR using the pPIC9K plasmid as a template (succeeded).
The sequencing results of the three samples pESC-TRP-Aga2-TFD-2/-4/-5 constructed by Gibson assembly are correct
Membrane Bio-Reactor Module
Pick a single yeast colony without gRNA plasmid from the SC+FOA plate and perform colony PCR verification.
August 27th
Yeast Surface Display Module
The four fragments of panARS-HZP vector, α-signal peptide, 6×His-TFD, and GS linker-Agα1 are recombined by the Gibson assembly to construct the panARS-HZP-α signal peptide-6×His-TFD-GS linker-Agα1 plasmid.
Membrane Bio-Reactor Module
Preserve and amplify the gRNA3-7 with the correct sequence.
Strains BY4741-pTEF1-FLO11 and BY4741 were amplified.
August 28th
Yeast Surface Display Module
Send successful E. coli colonies to sequence.
Transformed Saccharomyces cerevisiae EBY100 using the lithium acetate method with the pESC-TRP-Aga2-TFD plasmid.
Membrane Bio-Reactor Module
Perform dry weight experimental test cultures
Conduct preliminary flocculation experiments and record changes in cell flocculation status.
Obtain comparative micrographs of cell-cell adhesion.
August 30th
Membrane Bio-Reactor Module
Measure OD600 values of BY4741 and BY4741 pTEF1-FLO11; plotting dry weight change curves
September
September 2nd
Protein Engineering Module
Design a pH gradient experiment to test the effect of different pH (pH=6.5~8) on protein adsorption of rare earth ions; the results show that TFD-M has a fluorescence value at low pH
Yeast Surface Display Module
Construct the panARS-HZP-TFD plasmid by Gibson ligation, electroporate it into Pichia pastoris, and culture it on plates at 30°C.
September 3rd
Yeast Surface Display Module
Measure the growth curves of yeast strains displaying TFD proteins on their surface in Tb3+ medium
September 5th
Protein Engineering Module
Use hydrochloric acid to directly prepare pH = 2/3/4/5/5.5/6 solution to continue testing the effect of low pH conditions on protein adsorption of rare earth ions
Yeast Surface Display Module
Measure protein surface display using flow cytometry in four strains of Saccharomyces cerevisiae: EBY100, EBY100 (pESC-TRP-Aga2), EBY100 (pESC-TRP-Aga2-TFD), EBY100 (pYD1-Aga2-TFD)
Preparation of BMGY and BMMY culture medium for inducing protein expression in Pichia pastoris
September 7th
Yeast Surface Display Module
Saccharomyces cerevisiae EBY100, EBY100 (pESC-TRP-Aga2), EBY100 (pESC-TRP-Aga2-TFD), and EBY100 (pYD1-Aga2-TFD) induce TFD surface display protein expression again
Pichia pastoris GS115 and Pichia pastoris GS115 transformed with panARS-HZP-TFD plasmid synchronously induce TFD surface display protein expression
September 13th
Yeast Surface Display Module
Measure the protein surface display of the following yeasts using flow cytometry: ① Saccharomyces cerevisiae: EBY100, EBY100 (pESC-TRP-Aga2), EBY100 (pESC-TRP-Aga2-TFD), EBY100 (pYD1-Aga2-TFD);
② Pichia pastoris: GS115, GS115 (panARS-HZP-TFD). All successful!