During the April to June, we collected the data, constructed the model and trained the model. By using our model, we predicted the high-expression synthesized promoter and began to test it by wet lab. We conducted the wet lab from July to November.
Wet Lab
Preliminary Experiment
Episomal single reporter system expressing a constitutive red fluorescent protein (mcherry) wasdescribed and was uesd to create plasmid.
Episomal dual reporter system expressing a constitutive red fluorescent protein (mcherry) and a variable yellow fluorescent protein (YeGFP) were described and were uesd to create plasmid.
The plasmids were stored at -20℃(the plasmids were introduced with the
AmpR gene and the URA3 gene)
Made 20 YPD culture medium added Amp
Made the yeast-competent cells and store at -80℃
7.14-7.20
Inserted the mcherry plasmid and mcherry+YeGFP plasmid into the INVSC1 yeasts
The INVSC1 yeasts were streaked with a disposable loop on the YPD solid medium(adding the amp) and grown at 30 °C for 72 h. Screen the colonies on the YPD solid medium with amp.
Made the YPD and Sc-Ura fluid nutrient medium and autoclaved it for 20 minutes.
After the yeast grown in single colony, picking out the single colony into the YPD fluid nutrient medium and putting into the 30℃ horizontal rotators.
During this time, yeasts failed to grow on some of the YPD fluid nutrient medium and we inserted the plasmid again.
7.21-7.27
Extracted the plasmids from the yeast and store it at -80℃.
After Diluted the yeast for four times in 48h, with the first and the second time using the YPD,while the third and the fourth time using the Sc-Ura.
After the OD600 reached 0.4-0.6, we can dilute it with new fluid nutrient medium.
7.28-8.3
Measured OD600 of this diluted yeast suspension. Centrifuged at 4 °C at 3,500 rpm for 7 min and washed it with PBS for two times , useing flow cytometry to reportthemcherry or the GFP expression signal
Made the slide using the INVSC1 yeasts cell suspension and then oberserved the slides under the fluorescent microscope.
Formal experiment
8.4-8.10
Created the plasmid with the episomal dual reporter system and the library ofDNA sequences was inserted within a promoter scaffold. We got a series of plasmid totally 20 plasmids.
Made 40 YPDA culture mediums added Amp
Cultivated the INVSC1 yeasts for three days and made the INVSC1 yeast-competent cells and store at -80℃
8.11-8.17
Inserted the plasmids into the INVSC1 yeast, all the yeastswere streaked with a disposable loop on the YPD solid medium and grown at 30 °C for 72 h.
Made the YPD and Sc-Ura fluid nutrient medium.
After the INVSC1 yeast grown in single colony, picking out the single colony into the YPD fluid nutrient medium and putting into the 30℃ horizontal rotators.
The INVSC1 yeasts which were inserted with H1, ADH plasmid failed to grow on the culture medium.
8.18-8.24
Diluted the INVSC1 yeasts for four times in 48h, the first and the second dilution used the Sc-Ura fluid nutrient medium while the third and the forth used YPDA.
Extracted the plasmids from the yeasts and store it at -80℃
During the dilution,the INVSC1 yeasts which were inserted with H2,H4,H6,H7,L3,TEF,LTB-eGFP were polluted.
We inserted these plasmids into the INVSC1 yeasts renewedly.
8.25-8.31
After inserting the plasmid renewedlly, incubating the plates at 30 degrees
Extracted all the plasmids from the targeted microorganism and stored it at -80℃ to backup
After diluted the INVSC1 yeast for four times, measuring OD600 of this diluted INVSC1 yeast suspension. Centrifuged at 4 °C at 3,500 rpm for 7 min and washed it with PBS for two times , useing flow cytometry to reportthemcherry and
GFP expression signal
LTB-yeGFP
9.1-9.7
Extracted all the plasmids from the targeted microorganism and stored it at -80℃ renewedly to get the synthesized promoter and the LTB-eGFP plasmid frame.
Using double enzymes to perform sequential DNA cleavage and ran the agarose gel to confirmand the PCR to amplify. Ligated the LTB-eGFP plasmid frame with the synthesized promoter and the nature promoter one by one.
9.8-9.14
After we got the synthesized plasmid, inserted these plasmids into the INVSC1 yeasts and cultivated with the YPD/YPDA fulid nutrient medium.
Extracted the synthesized plasmids from the yeasts and store it at -80℃ to backup.
During the extraction, we failed to extract the plasmids and we extracted renewedly. Made 40 YPDA culture mediums added Amp.
Made the YPD and Sc-Ura fluid nutrient medium and autoclaved it for 20 minutes.
9.15-9.21
The INVSC1 yeasts with synthesized plasmids were streaked with a disposable loop on the YPD solid medium(adding the amp) and grown at 30 °C for 72 h. Screen the colonies on the YPD solid medium with amp. Each sample took four single conoly and cultivated individually with four 50ml centrifuge tube.
Diluted the INVSC1 yeasts with synthesized plasmids for four times, measuring OD600 of this diluted INVSC1 yeast suspension. Centrifuged at 4 °C at 3,500 rpm for 7 min and washed it
with PBS for two times , useing flow cytometry to reporttheLTB-eGFP expression signal.
9.22-9.28
Extract the total RNA from the INVSC1 yeasts (totally 50 samples)and stored for QRT-PCR.
Extract the total protein from the INVSC1 yeasts(totally 30 samples and stored at -80℃)
Ran the SDS gel of the INVSC1 yeasts and chose a suitable WB internal reference.
9.29-11.5
We used the Gapdh as the internal reference and ran the SDS gel, we also ran the Q-PCR to detect the gene expression.