Experiments

Copy and combine the gene fragments required by the project (BAT2-L-TEF1-ATF1-CYC1-TEFp-NrsR-TEFt-BAT2-R); add homology arms (BAT2-L&BAT2-R); and assemble the fragments for restriction enzyme digest and ligation (T4). The sequence map of the fragments is shown in fig1.1-1.

The BAT2 fragment codes for a key enzyme that leads to the production of higher alcohols during the brewing process. Therefore, in order to control the metabolism of higher alcohols in yeast, we used homologous recombination to replace the BAT2 with DNA strand TEF1-ATF1-CYC1-TEFp-NrsR-TEFt. Among them, the ATF1 fragment codes for an enzyme that can acetylate long-chain fatty alcohols and improve the taste of brewed products, and NrsR will be used in later experiments to screen the yeast cells that are modified from ones that are not.

Among them, the homology arms on both sides need to be extracted and amplified from the yeast genome template BAT2, and the four fragments of CYC1-TEFp-NrsR-TEFt need to be extracted and amplified from the yeast genome template pH Cas9.

Fig1.1-1 The figure shows the target fragments, with the promoters, terminators, upstream and downstream homology arms, primers and enzymes labeled.

1.2.1 Objective

The five fragments of BAT2-L, TEF1, ATF1, CYC1-TEFp-NrsR-TEFt, and BAT2-R were copied for subsequent assembly.

1.2.2 Materials

  1. PCR Amplifier
  2. 0.2 mL PCR tubes
  3. PrimeSTAR 2X (a conbination of primer, DNTP, buffer)
  4. primer (CYC1-F, TEFt-R, BAT2-R-U-F, BAT2-R-U-R, TEF1p-F, TEF1p-R, ATF1-F, ATF1-R, BAT2-L-U-F, BAT2-L-U-R)
  5. Gene segments (BAT2-L, TEF1, ATF1, CYC1-TEFp-NrsR-TEFt, BAT2-R)
  6. ddH2O
  7. Dimethyl Sulfoxide(DMSO)

1.2.3 Procedure

1.2.3.1 System

The table 1.2.3.1-1 presents our system. The volume that we used was 20μl.

table 1.2.3.1-1: The table above demonstrates the system needed in corresponding PCR experiments for each part of the gene.

(The Saccharomyces cerevisiae genes in the table were from fresh yeast treated with 50 μl yeast lysate at 80°C for 10mins)


1.2.3.2 Condition/PCR program settings

The condition of the experiment is shown in table 1.2.3.2-1.

table 1.2.3.2-1: The table shows conditions of the first PCR for gene amplification

1.3.1 Objective

Assemble the five BAT2-L, TEF1, ATF1, CYC1-TEFp-NrsR-TEFt, BAT2-R fragments that were replicated in the previous step into the desired BAT2-L-TEF1-ATF1-CYC1-TEFp-NrsR-TEFt-BAT2-R segment and amplify.

1.3.2 Materials

  1. PCR Amplifier
  2. 0.2 mL PCR tubes
  3. PrimeSTAR 2X (a conbination of primer, DNTP, buffer)
  4. primer (CYC1-F, TEFt-R, BAT2-R-U-F, BAT2-R-U-R, TEF1p-F, TEF1p-R, ATF1-F, ATF1-R, BAT2-L-U-F, BAT2-L-U-R)
  5. Gene segments (BAT2-L, TEF1, ATF1, CYC1-TEFp-NrsR-TEFt, BAT2-R)
  6. ddH2O
  7. Dimethyl Sulfoxide(DMSO)

1.3.3 Procedure

1.3.3.1 System

The second PCR system involves combining all the five targeted sequences from the previous step together after being extracted from the two yeast genome templates BAT2 and pH Cas9. In this case, we directly combined the five gene sections using the table 1.3.3.1-1.The volume we uses was still 20 microliters.

To ensure successful assembly, we used dimethyl sulfoxide, or DMSO for short. DMSO is an organosulfur compound with a high polarity and high dielectric constant, and used in PCR to disrupt secondary structure formation in the DNA template.

table 1.3.3.1-1 This table illustrates the amounts of reagents used for our assembly.

1.3.3.2 Condition/PCR program settings

The table 1.3.3.2-1 shows the second PCR's condition. The durance and temperature for the annealing stage and extension stage were changed to match our purpose of combining the five strands.

The probability of successfully connecting the five gene fragments hardly reaches 1%, so we increased the number of loops to increase the probability.

table 1.3.3.2-1 This table shows the condition of the second PCR.

After each PCR process, we need to verify the success of the experiment, perform analysis, and recover the useful fragments.

Gel electrophoresis is done at 150v for 20 minutes, and put into the gel UV instrument for verification, analysis and photographing records.

After photographing and analyzing, we cut out the gel containing the DNA that are correctly replicated and extract the DNA from the gel. The gel recovery reagent we use is the DiaSpin DNA Gel Extraction Kit produced by Shanghai Sangon Biotech Co., Ltd. Please refer to the product manual on the official website of Sangon Biotech for detailed operating instructions.Generally, after recovery, we will also detect the nucleic acid concentration of the fragments and record them.

Obtain yeast competent cells by changing the permeability of the cell membrane through the lithium acetate transformation method, and transfer the PCR amplified gene fragment BAT2-L-TEF1-ATF1-CYC1-TEFp-NrsR-TEFt-BAT2-R into yeast cells to obtain Saccharomyces cerevisiae A (SF-1).

  • PEG3350(50%)
  • LiAc(1M)
  • ssDNA(2g/mL)
  • BAT2-L-TEF1-ATF1-CYC1-TEFp-NrsR-TEFt-BAT2-R gene fragment
  1. Pick up the saccharomyces cerevisiae into the 5ml YEPD culture medium, and grow overnight.
  2. Transfer the YEPD culture medium into 50ml YEPD (30℃, 240rpm) until OD600 absorbance value is 0.8-1.0 (4-5h).
  3. At 20℃ (3000g, 5min) centrifuge and repeat.
  4. Add 1ml sterile water, resuspend into 1.5ml EP tube to collect the saccharomyces cerevisiae (1200g,30s).
    *Saccharomyces cerevisiae competent have prepared.
  5. Meanwhile put 50μl ssDNA into 96℃ water bath for 5min.
    (then put them on ice immediately)
  6. Centrifuge 100μl saccharomyces cerevisiae (12000g, 30s). Then Throw supernatant away.
  7. Add 240μL PEG3350, 36μL LiAc, 50μL ssDNA into a 1.5ml EP tube and then mix.
  8. Add all materials that have been prepared at beginning into yeast competent state (42℃ water bath,15min).
  9. Centrifuge again (12000g, 30s), throw supernatant away, then add 200μl saccharomycete solution on YEPD board (200ug/ml) 30℃ store in Biological incubator.

Construct the pYES2-ATF1 recombinant plasmid and transform into Saccharomyces cerevisiae A.

The pYES2-ATF1 recombinant plasmid (plasmid A) was constructed, and it was transformed into DH5a competent cells and screened by ampicillin resistance. After verification, the pYES2-ATF1 recombinant plasmid was extracted from the bacteria and transformed into Saccharomyces cerevisiae A to produce Saccharomyces cerevisiae B (SFA-1).

The ATF1 gene expression cassette (ATF1-cas-F, ATF1-cas-R) was obtained during the amplification of the BAT2-L-TEF1-ATF1-CYC1-TEFp-NrsR-TEFt-BAT2-R gene fragment. The above PCR fragments were double digested with Spe1 and Sal and recovered. The pYES2-gRNA-hyg-MCS vector were also double digested with Spe1 and Sal1 and revocerd. Use ligase to ligate the double-digested vector and the PCR fragment. The ligation was transformed into E. coli for ampicillin resistance screening.

3.2.2.1 Objective

  1. Extract the pYES2 plasmid for subsequent double digestion.

3.2.2.2 Materials

  • Centrifuge
  • 1.5 mL micro-centrifuge tubes
  • DiaSpin Plasmid Mini-Preps Kit

3.2.2.3 Procedure [1]

  1. Column equilibration: Place a Spin Column in a clean collection tube, add 500 μl Buffer S to column. Centrifuge for 1 min at 12000g in a table-top microcentrifuge. Discard the flow-through, and set the Spin Column back into the collection tube.
  2. From the overnight culture, transfer 1.5-5 ml bacteria solution into a microcentrifuge tube. Harvest the bacterial cells by centrifugation at 8000g for 2 min, then remove all supernatant.
  3. Re-suspend the bacterial pellet in 500 μl Buffer SP1 (Ensure that RNase A has been added to Buffer SP1).
  4. Add 250 μl Buffer SP2 and mix thoroughly by inverting the tube 5-10 times, let stand for 2-4 min at room temperature.
  5. Add 350 μl Buffer SP3 and mix immediately and thoroughly by inverting the tube 5-10 times.
  6. Centrigufe for 5-10 min at 12000g in a table-top microcentrifuge. Carefully transfer the supernatant to the spin colume (note: do not touch the precipitate, and do not shake the tube when taking it out of the centrifuge). Centrifuge for 30 sec at 8000 xg. Discard the flow-through and set the spin column back into the collection tube.
  7. Wash the Spin Column by adding 500 μl Wash Solution (ensure the anhydrous alcohol has been added to the wash solution) and centrifuging for 30 sec at 9000g. Discard the flow-through and set the Spin Column back into the Collection Tube. (Repeat once)
  8. Centrifuge the empty collection tube for an additional 1 min at 9000 xg to remove residual Wash Solution.

Place the Spin Column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50-100 μl Elution Buffer to the center of the Spin Column, let stand for 1 min at room temperature, and centrifuge for 1 min at 9000g. Preserve the DNA solution in the tube.


[1] https://www.protocols.io/view/plasmid-extraction-bndrma56?step=8

3.2.3.1 Objective

The ATF1 gene expression cassette is a combination of ATF1, CYC1, and TEF1 fragments. In Saccharomyces cerevisiae B, this gene cassette is to add an additional ATF1 fragment in addition to Saccharomyces cerevisiae A. This can better enhance the taste of the brewed product. The sequence of the ATF1 gene expression cassette is shown in Fig 3.2.3-1.

Fig 3.2.3-1 Shows the sequence of the ATF1 gene expression cassette.

3.2.3.2 Materials

  • PCR Amplifier
  • 0.2 mL PCR tubes
  • PrimeSTAR 2X (a conbination of primer, DNTP, buffer)
  • primer (ATF1-cas-F, ATF1-cas-R)
  • Gene segments (TEF1, ATF1, CYC1)
  • ddH2O
  • Dimethyl Sulfoxide(DMSO)

3.2.3.3 Procedure

3.2.3.3.1 system

Like other assembly in the experiment, the assembly of the ATF1 gene expression cassette also uses a 20 μl system.

To see the TEF1, ATF1, CYC1 copying protocol, please refer to section 1.2.3.

See Table 3.2.3.3-1 for the volume of reagents added to the system.

Table 3.2.3.3-1 shows the capacity of reagents added to the system.

3.2.3.3.2 Condition/PCR program settings

Please go back to section 1.3.3.2 for reference.

3.2.4.1 Objective

Double digestion ( SpeI and SalI ) digests a DNA substrate with two restriction endonucleases simultaneously, is a common timesaving procedure.[1]

In this experiment, two restriction endonuclease sites locate at the 1026bp and 1452bp in the pYES2 plasmid, and the two enzymes will cut the SNR52 promoter, gRNA scaffold and SUP4 terminator into a fragment in a total of 424bp. The fragment was excised to ensure that the excised gap allowed ligation of the ATF1 gene expression cassette to form the pYES2-ATF1 plasmid.

Fig 3.2.4.1-1 and Fig 3.2.4.1-2 are the sequences of the two restriction enzymes for reference.


Fig 3.2.4.1-1 shows the sequences of Spe I restriction endonuclase.[2]
Fig 3.2.4.1-2 shows the sequences of Sal I restriction endonuclease.[3]

3.2.4.2 Materials

  • pYES2 plasmid (31ng/μl)
  • Restriction Endonuclease
    • Spe I
    • Sal I
  • Cutsmart buffer (10x)
  • ddH2O
  • SFA-1 strain

3.2.4.3 Procedure

  1. Add 32.3 μl of the pYES2 plasmid, which has not been digested, to a 10 ml centrifuge tube.
  2. Add 1 μl of each of spe I and Sal I restriction enzymes. Then add one microliter of Cutsmart buffer.
  3. Add 10.7 µl of ddH2O.
  4. Place the existing solution in a 37-degree incubator for one hour to complete the digestion.

[1] https://www.genscript.com/what-is-dna-ligation.html

[2] https://international.neb.com/products/r0133-spei#Product%20Information.

[3] https://www.neb.sg/products/r0138-sali#Product%20Information.

3.2.5.1 Objective

DNA ligase catalyzes the formation of two covalent phosphodiester bonds between the 3’ hydroxyl group of one nucleotides and the 5’ phosphate group of another in an ATP dependent reaction. In molecular cloning the ligation reaction follows the digestion of the gene insert and the target vector.

In this experiment, we will connect the ATF1 gene expression cassette with the already digested pYES2 plasmid to obtain the pYES2-ATF1 plasmid.

3.2.5.2 Materials

  • pYES2 plasmid (enzymatically digested)
  • ATF-1 gene expression cassette
  • Nourseothricin resistance gene
  • T4 DNA Ligase Reaction buffer
  • T4 DNA Ligase
  • ddH2O
  • incubator

3.2.5.3 Procedure

The system is 20 μl.

  1. Add 3 μl of pYES2 plasmid that has been digested.
  2. Add 1 μl of each of the ATF1 gene expression cassette and the nourseothricin resistance gene.
  3. Add 1 μl of T4 DNA Ligase and 1 μl of T4 DNA Ligase Reaction buffer.
  4. Add 12 μl of ddH2O.
  5. The solution was placed in an incubator at 35 degrees for 30 minutes (alternatively, two hours at room temperature). Allow the enzymes to react and ligate.
  6. After the connection is completed, put it in the minus twenty degree refrigerator for freezing.

[1] https://www.genscript.com/what-is-dna-ligation.html

3.2.6.1 Objective

Transform plasmid A into DH5a competent cells and screen them by ampicillin resistance.

3.2.6.2 Materials

  • Water bath
  • Centrifuge
  • incubator shaker
  • ammoniacal plate
  • 1.5ml micro-centrifuge tubes
  • plasmid A
  • DH5α gene
  • LB culture medium

3.2.6.3 Procedure

  1. Add 20 ul plasmid A and 100 ul DH5α
  2. Place on ice to incubation (30 minutes)
  3. 42 ℃ heat stroke for 90 seconds
  4. Place in ice again for 2-3 minutes
  5. Add 700μl LB 37 ℃ for 1 hour in 220 rpm incubator shaker
  6. Inoculate on an ammoniacal plate
  7. choose postive clone to sequencing

3.2.7.1 Objective

Extract the plasmid from the screened E. Coli, where we get our target plasmid--plasmid B.

3.2.7.2 Materials

Please go back to part 3.2.2.2 for reference.

3.2.7.3 Procedure

Please go back to part 3.2.2.3 for reference.

3.3.1 Objective

Plasmid A was transformed into Saccharomyces cerevisiae A containing the BAT2-L-TEF1-ATF1-CYC1-TEFp-NrsR-TEFt-BAT2-R gene fragment by lithium acetate transformation.

3.3.2 Material

Please go back to section 2.2 for reference.

3.3.3 Procedure

Please go back to section 2.3 for reference.

3.4.1 Importance and uses

Whether it is our constructed Saccharomyces cerevisiae strain or wild-type Saccharomyces cerevisiae strain. All need an environment for growth, and the culture medium is widely used in scientific research as the environment for these kinds of saprophytic organism.

In our experiment, the two types of culture medium are used to determine the growth curve of each Saccharomyces cerevisiae strain and the fermentation test in lab by stimulating winery.

3.4.2 Ingredients

3.4.2.1 LB

System: 100mL

  • Yeast extract: 1 g
  • NaCl: 1 g
  • Trytone: 2 g
3.4.2.2 2%YEPD

System: 200mL

  • Yeast extract: 2 g
  • Trytone: 4 g
  • Glucose: 4 g

3.4.3 Use in application scenarios

3.4.3.1 Determine the growth curve of Saccharomyces cerevisiae strain
  • Add Strain A (SF-1), Strain B (SFA-1) and wild type strain (WT) 125 μl for each strain into the 12.5 ml YEPD culture medium, respectively, seal the medium then let it stand in a incubator at 30°C, and take out the fungus solution at the corresponding time and determine the absorbance.
3.4.3.2 Fermentation test in lab by stimulating winery
  • Add 2 ml Strain A (SF-1), Strain B (SFA-1) and wild type strain (WT) into the 20 ml 16% YEPD culture medium, respectively, and seal the medium then let it stand in a incubator at 30°C for 60 hours. After the fermentation, determine the content of higher alcohol.

4.1 Objective

The purpose of this test is to test whether our constructed S. cerevisiae strain can reach the level of wild-type S. cerevisiae strain.

The test is based on three strains: SF-1, SFA-1, and wild-type, with data acquired between hours, allowing the target microorganism to grow and test the OD600 absorbance of each culture by using the spectrophotometer, at each node Absorbance was measured 9 times, three averages were taken, and the growth curve was drawn using R language.

4.2 Material

  • spectrophotometer
  • shaker
  • R software
  • Cuvette
  • 10ml centrifuge tube
  • SF-1, SFA-1, and WT strains
  • distilled water

4.3 Procedure

Experiments were carried out at 2, 4, 7, 16, 24, 32 hours after the strain started growing.

  1. Take out the three strains from the shaker, extract 1.5 ml of each bacterial solution in the ultra-clean bench and pour it into a clean 10-ml centrifuge tube.
  2. Before testing the OD600 absorbance of the strain, inject distilled water into the cuvette and calibrate the spectrophotometer.
  3. Inject the subpackaged bacterial solution into the cuvette, put it into a spectrophotometer to detect and record the OD600 absorbance, and test each bacterial solution nine times. After each test, calibrate the spectrophotometer using a cuvette filled with distilled water.
  4. Screen the tested OD600 absorbance data, screen 3 medians of each bacterial solution at each checkpoint, and write them into table 4.3-1 for image generation. Table 4.3-1, Raw data obtained at each time period 2, 4, 7, 16, 24, 32 hours respectively. Each strain WT, SF-1, SFA-1 was tested with Spectrophotometer three times, to obtain OD600 absorbance level.
    Time (h)/Type SF-1 SFA-1 WT
    2 0.076 0.052 0.016
    4 0.106666667 0.108 0.049
    7 0.18 0.320333333 0.109
    16 1.007333333 1.732333333 0.639
    24 2.558333333 2.517666667 1.105
    32 2.554666667 2.528333333 1.233666667
  5. After all the data were recorded, use R software to draw the image of the growth curve, This diagram uses LOG2 to fit the curve and we calculated the p-values when comparing with WT using the ANOVA Test as three groups were considered. Figure 4.3-1 is the generated graph 's source code.
    Fig4.3-1 The figure shows the source code that we use to draw the curve. The text after the # symbol is the comment for the code.

5.1 Objective

Fermentation testing of yeast has been around for a long time. In this experiment, the test was mainly to verify whether the SFA-1 and SF-1 S. cerevisiae strains did not produce higher alcohols during fermentation compare to wild type strain.

5.2 Material

    YPD culture medium
  • Three Saccharomyces cerevisiae strains (SFA-1, SF-1, WT)
  • Simulated alcohol fermentation medium
  • high-performance liquid chromatography
  • Chromatographic and capillary columns

5.3 Procedure

  1. Three of the overnight cultures in YPD medium were inoculated into simulated alcohol fermentation medium at a concentration of 1:10.
  2. The simulated alcohol fermentation medium was allowed to stand at 30 degrees for 60 hours to ferment. Each medium was weighed every 12 hours.

When the fermentation is over, the higher alcohol content is determined. Each strain needs to extract the higher alcohol content data three times, and then calculate the average value for each strain. Chromatography was performed by other laboratories due to hardware limitations.

  1. Using distillation, the fermentation broth is filtered away, leaving the strains for high-performance liquid chromatographic analysis.
  2. Send it to a testing agency to use high-performance liquid chromatography to detect the concentration of higher alcohols of strians.
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