April,1
Experiment basic operation exercise, promoter PCR attempt.
May,1
gRNA construction, promoter PCR attempt.
Jane,1
harmOR10 gene integration attempt, promoter FUS1 PCR.
July,9
Related experiments of reporter plasmid construction;
Related experiments of harmOR10 gene integration（Culture BY4741 yeast seed solution）.
July, 10
Related experiments of reporter plasmid construction (The promoter, plasmid skeleton and mCherry fragment were
digested respectively, and the gel was cut and recovered);
Related experiments of harmOR10 gene integration（The seed liquid was transferred to a shake flask and the
sensitive state was prepared after 5h）.
July,13
Related experiments of reporter plasmid construction (the scribe plate grows more colonies, and verify the primer
synthesis);
Related experiments of harmOR10 gene integration（Competent preparation was completed and BY4741 seed solution was
inoculated）;
Experiment of Schizonepeta alcohol fermentation（activation of strain bank strains (3-1 / 3-2 / 3-3 / 3-4)）.
July,14
Related experiments of reporter plasmid construction (redesigned the primers and performed PCR again, observe the
results);
Related experiments of harmOR10 gene integration（Continue to raise yeast (grow into single colonies of appropriate
size in solid medium for 3 days);
Experiment of Schizonepeta alcohol fermentation (Transfer to shake flask; Preserve the bacteria and inoculate into
the peptonea tube (seed solution culture)).
July,15
Related experiments of reporter plasmid construction (FUS1 and mCherry plasmids were tested, greemix pcr50 degree
was 1.5min, and there was almost no band after gel running, It may be necessary to perform the ligation
transformation of FUS1 and mCherry again, or re inoculate the plate.);
Experiment of Schizonepeta alcohol fermentation (Transfer to galactose medium).
July,16
Related experiments of reporter plasmid construction (PCR was performed on mcerry and FUS1 that had not been
enzymatically digested. There were bands in the gel. PCR was recovered, and the DNA concentration was measured. It
was placed in the refrigerator at - 20 degrees; FUS1 amplification failed and was discarded; Digested mCherry and
FUS1 with 10 μ LT4 ligase enzyme ligation system enzyme ligation, inoculate amp plate and put it in 37 °
incubator; PCR 4 tubes of FUS1);
Experiment of Schizonepeta alcohol fermentation (Transfer to galactose medium and ferment for two days).
July,17
Related experiments of reporter plasmid construction (Enzymatic ligation, transformation and inoculation).
Related experiments of harmOR10 gene integration（gRNA plasmid skeleton extraction, gRNA annealing, GRNs plasmid
skeleton digestion）.
July,18
Related experiments of reporter plasmid construction (Checkpoint, FUS1 and mCherry enzyme linked, transformed,
coated and inoculated);
Related experiments of harmOR10 gene integration（Enzyme linked, transformation）;
Experiment of Schizonepeta alcohol fermentation (GC, Sampling, extraction, sample preparation and storage in
refrigerator).
July,19
Related experiments of harmOR10 gene integration（Enzyme linked, grna2 and grna3 fragments were enzymatically
linked to the plasmid backbone and transformed into E. coli）
July,20
Related experiments of reporter plasmid construction (Two primers were used to check the spots respectively, and
both failed);
Related experiments of harmOR10 gene integration（Plasmids were extracted, sequenced, and bacteria were preserved.
gRNA2, 3 test points, and the test points were successful; Amplification of gRNA2, 3）;
Experiment of Schizonepeta alcohol fermentation (Transfer into 30ml shake flask (glucose medium, culture seed
liquid))
July,21
Related experiments of reporter plasmid construction (Plasmid extraction, bacteria preservation, sequencing; Check
point: the two primers are mixed and the check point is successful; Select points and expand cultivation.);
Related experiments of harmOR10 gene integration（transformation after sequencing）;
Experiment of Schizonepeta alcohol fermentation (Transfer to galactose medium, transfer bacteria 1 and 2 to six
shake flasks (glucose medium)).
July,22
Related experiments of reporter plasmid construction (Sequencing, Plasmid Extraction, concentration determination
and recording);
Related experiments of harmOR10 gene integration（Construct yeast competent state and put it in 4 ℃ refrigerator）;
Experiment of Schizonepeta alcohol fermentation (The two groups of bacteria were centrifuged together with the
culture medium, and then the precipitates were washed twice with sterile water, transferred to galactose culture
medium, and cultured in a shaking table at 30 ℃).
July,24
Related experiments of harmOR10 gene integration（Transformation (grna2, 3 and whether to add donor, four groups in
total)）;
Experiment of Schizonepeta alcohol fermentation (Sampling: take out the triangular flask from the shaker, add 1ml
of bacterial solution into 2ml centrifuge tube, add 1ml of ethyl acetate into each tube, mix it with a vortex for
30s, shake it on the shaker for 40min, mix it again, shake it for 40min, and take the upper layer.).
July,25
Experiment of Schizonepeta alcohol fermentation (Prepare YPD (A));
Transformation of Schizonepeta alcohol fermentation strain (Inoculate gRNA plasmid bacteria and plasmid bacteria
containing the gene to be introduced).
July,26
Experiment of Schizonepeta alcohol fermentation (3 and 4 were inoculated into test tubes and cultured in a
shaker);
Transformation of Schizonepeta alcohol fermentation strain (Plasmid extraction, gRNA extraction, put in - 20
degree refrigerator.).
July,27
Related experiments of harmOR10 gene integration（Pick points, break cells, PCR, glue run, fail）;
Experiment of Schizonepeta alcohol fermentation (Bacteria 3 and 4 were inoculated into six culture media and
cultured in a shaker at 30 ℃ for 24h);
Transformation of Schizonepeta alcohol fermentation strain (Pick points, break cells, PCR, glue run, fail.).
July,28
Related experiments of harmOR10 gene integration（Cell breakage, PCR checkpoint, PCR recovery. The result is not up
to standard and is discarded.）;
Experiment of Schizonepeta alcohol fermentation (Transfer to galactose medium and culture for 24h).
July,30
Experiment of Schizonepeta alcohol fermentation (extraction).
August,1
Related experiments of reporter plasmid construction (Promoter PCR);
Related experiments of harmOR10 gene integration（Checkpoint (phantase)）;
Experiment of Schizonepeta alcohol fermentation (GC-MS chromatography).
August,2
Related experiments of reporter plasmid construction (pcr fus1);
Related experiments of harmOR10 gene integration（pcr donor，）;
August,3
Related experiments of reporter plasmid construction (Yeast plasmid with mCherry was extracted, digested, purified
and recovered, linked with FUS1 two fragments, transformed in large intestine and inoculated);
Related experiments of harmOR10 gene integration（Construction, transformation and inoculation of competent yeast）;
Experiment of Schizonepeta alcohol fermentation (Seed liquid, inoculation);
Transformation of Schizonepeta alcohol fermentation strain (Construction, transformation and inoculation of
competent yeast).
August,4
Related experiments of reporter plasmid construction (Test point, inoculation at point 8, culture for 12-16h);
Experiment of Schizonepeta alcohol fermentation (Transit glucose medium).
August,5
Related experiments of reporter plasmid construction (Bacteria preservation, sequencing and plasmid extraction.);
Experiment of Schizonepeta alcohol fermentation (Transfer from glucose medium to galactose medium and incubate for
48 hours);
August,6
Related experiments of harmOR10 gene integration（At the test point, the yeast is broken (boiling solution, 20ul,
98 ℃, 30min). After boiling, the yeast is centrifuged, and the supernatant is taken for PCR with phantase.）;
Transformation of Schizonepeta alcohol fermentation strain (Jl1491 plasmid was extracted and PCR (primers: cyqr,
ADHF)).
August,7
Experiment of Schizonepeta alcohol fermentation (Extraction, GC);
Transformation of Schizonepeta alcohol fermentation strain (Take the plasmid of jl1491 gRNA, transform it into
yeast am3-2, and coat the plate).
August,8
Related experiments of harmOR10 gene integration（Plasmid extraction, PCR, transformation, plating）;
Transformation of Schizonepeta alcohol fermentation strain (PCR JL1491, transformation, plating).
August,11
Related experiments of harmOR10 gene integration（The phenomenon of paste plate (too dense colonies) appeared in
transformation, because the cas9 gene contained in the wild-type BY4741 strain used does not match the gRNA）;
Experiment of Schizonepeta alcohol fermentation (Spectrum observation: in the last batch of fermentation
experiments, all strains except 3-1 had product peaks, of which 3-2 and 3-4 products were relatively more);
Transformation of Schizonepeta alcohol fermentation strain (Pick and check points).
August,13
Related experiments of harmOR10 gene integration（Plasmid extraction, PCR, transformation）;
Transformation of Schizonepeta alcohol fermentation strain (Plasmid extraction, PCR, unsuccessful, re
inoculation).
August,14
Transformation of Schizonepeta alcohol fermentation strain (Plasmid extraction, PCR, transformation, culture).
August,16
Related experiments of harmOR10 gene integration（Two colonies grow out of the ste plate - all the test points have
correct bands, and the PCR products are sent for testing）;
August,17
Integration of reporter plasmid and harmOR10（Report plasmid PCR, gel run test, recovery, concentration
measurement, preservation; Ste-harmor10 1 / 2 each preserved bacteria, shake the bottle for 5h, prepare the
sensitive state, store it at 4 ℃, and collect the sequencing results of harmor10 gene. The results are correct
compared with the original genome）；
August,18
Integration of reporter plasmid and harmOR10（P426 plasmid was extracted; Ste-harmor10 transformation）；
August,19
Integration of reporter plasmid and harmOR10（The p426-21 plasmid was extracted, the concentration was measured,
and stored）；
Transformation of Schizonepeta alcohol fermentation strain (Prepare 3-2 / 3-4 competent state; Jl1491
vaccination).
August,20
Transformation of Schizonepeta alcohol fermentation strain (Jl1491 inoculation is still not long; Use JL1491 on
the amp-lb plate coated a few days ago, pick a spot, PCR, failed).
August,21
Integration of reporter plasmid and harmOR10（Both 2-2 and 1-2 grow a colony, pick a spot to verify (phanta PCR),
run the glue, and the band is about 1500 ∓500）；
Transformation of Schizonepeta alcohol fermentation strain (Plasmid PCR, gel run verification).
August,23
Transformation of Schizonepeta alcohol fermentation strain (PCR with KOD one enzyme).
August,26
Integration of reporter plasmid and harmOR10（Re check the spot and take photos, but it does not run out of the
strip）；
Transformation of Schizonepeta alcohol fermentation strain (Reconvert with 3-2 competent state and inoculate).
Benzaldehyde fluorescence concentration detection (Ste-harmOR10-2 strain was amplified and cultured in
benzaldehyde with concentration gradient for 1 day).
August,27
Benzaldehyde fluorescence concentration detection (Fluorescence concentration detection; No regular results were
found).
August,28
Experiment of Schizonepeta alcohol fermentation (3-2 redo competent state, transformation).
August,29
Transformation of Schizonepeta alcohol fermentation strain (there are two points on the 8.23 board, and there are
no results; The plasmid of p426 was re extracted).
Benzaldehyde fluorescence concentration detection (Medium with gradient benzaldehyde concentration was transferred
and cultured for one day).
August,30
Transformation of Schizonepeta alcohol fermentation strain (There were many colonies in both plates).
Benzaldehyde fluorescence concentration detection (Fluorescence concentration and biomass were detected, but no
regular results were presented).
August,31
Transformation of Schizonepeta alcohol fermentation strain (Test points (8 for each plate, boiling,
centrifugation, phanta PCR, glue running) showed that there were bands around 2000, which were 3, 8 for 1 and 1, 2
and 4 for 2; Select 1-8 and 2-2 for sequencing and then connect with seed liquid for amplification for one day).
September,1
Experiment of Schizonepeta alcohol fermentation (Mlpla transfer strain fermentation experiment: transfer the seed
liquid into glucose YPD (a) shake flask).
Transformation of Schizonepeta alcohol fermentation strain (Mlpla gene integration sequencing was successful, and
the seed liquid was ready to integrate the neps1 gene, and then the Neps1 plasmid bacteria)
September,2
Experiment of Schizonepeta alcohol fermentation (Transfer to galactose medium).
Transformation of Schizonepeta alcohol fermentation strain (Neps1 donor PCR and integration).
September,3
Experiment of Schizonepeta alcohol fermentation (Sampling (24h)).
September,4
Experiment of Schizonepeta alcohol fermentation (Sampling (48h)).
September,5
Experiment of Schizonepeta alcohol fermentation (Seed liquid was inoculated (strains 1 and 2 after neps1
integration, and strain 1 without neps1 integration was used as the control group)).
Transformation of Schizonepeta alcohol fermentation strain (To verify whether neps1 insertion was successful, two
colonies were selected for sequencing).
Plasmid loss of Schizonepeta lactone strain (Inoculated strain).
Discussion with our PI (The self-contained promoter of ste2 is not strong enough, and the donor integrates the gal
promoter into the site; Positive control: keep ste2 gene, only do reporter gene integration, and test whether FUS1
promoter is effective; Fermentation experiment: compare the integrated neps1 strain and the non integrated neps1
strain, and observe the catalytic efficiency of neps1. If the catalytic efficiency is low, consider making
multiple copies).
September,6
Harmor10 gene integration (New) (Inoculate harmor10 plasmid bacteria and by4741-cas9 yeast).
Experiment of Schizonepeta alcohol fermentation (Seed liquid transfer).
Plasmid loss of Schizonepeta lactone strain (Inoculate the bacterial solution of Schizonepeta lactone No. 1 and
No. 2 cultured for 24 hours into the new test tube at the ratio of 1%).
Preparation of positive control bacteria (Inoculation of reporter plasmid and BY4741-cas9).
September,7
Experiment of Schizonepeta alcohol fermentation (After washing, the strain was connected to YPD medium and
cultured in a 30 ° shaker for 24h).
Plasmid loss of Schizonepeta lactone strain (Inoculate the bacterial solution of Schizonepeta lactone No. 1 and
No. 2 cultured for 24 hours into the new test tube at the ratio of 1%).
September,8
Experiment of Schizonepeta alcohol fermentation (Sampling and preparation of fermentation broth and storage in -
20 ℃ refrigerator).
Plasmid loss of Schizonepeta lactone strain (Strain 1 and strain 2 were connected to the plate by scribing and
placed in a 30 degree incubator).
September,10
Losing plasmid of schizonepeta tenuifolia lactone strain (Pick up the single colony (6 in total) on the plate and
mix it with sterile water, and apply it to YPD-G418 plate, SE-URA-G418 plate and SE-HIS-G418 plate respectively.)
Preparation of positive control bacteria (Positive control bacteria sampling verification, fail.)
September,11
Preparation of HarmOR10-GAL strain (HarmOR10-GAL plate spot selection verification, sending for testing (No. 5 and
No. 7 bacteria), and seed liquid culture.)
September,12
Transformation of HarmOR10-GAL strain (Competent preparation, fus1-mCherry gene integration.)
Preparation of positive control bacteria (Competent preparation, fus1-mCherry gene fragment integration.)
September,15
Fermentation experiment (Seed liquid inoculation (NEPS1 introduced strain))
September,16
Fermentation experiment (Seed liquid transfer)
September,17
Fermentation experiment (Transfer to galactose medium)
September,18
Fermentation experiment (Sampling, sample preparation, GCMS detection)
September,22
Fermentation experiment (Inoculation of seed liquid (starting strain, MLPLA modified strain))
September,23
Fermentation experiment (Seed liquid transfer)
September,24
Fermentation experiment (Transfer to galactose medium)
September,25
Fermentation experiment (Sampling, sample preparation, GCMS detection)