Logs

Detailed logs for the experiment:

Date	Works
8.5~8.6	Amplification of the target gene TPS1.
8.7	Ligate the pYET plasmid containing ADH2p and the TPS1 gene by ligase. Introduce into the receptor E. coli DH5-α for plate coating.
8.8	Colonies appeared in overnight culture, E. coli monoclonal clones were picked and made into suspension, partially amplified for one day and partially lysed for PCR verification. PCR verification was successful.
8.9	Use the E. coli suspension amplified for one day, extract the plasmid, carry out enzyme digestion to verify plasmid construction is successful.
8.10	The plasmid was transferred into the YNB medium of the sensory yeast S288C and cultured for 48 hours for the emergence of single clones.
8.12	After 4 days of amplification, tryptophan-deficient flat had complete colonies, half of a single clone was picked for PCR verification, and the other half of the single clone that was successfully verified was transferred into 10 ml of pre-culture solution for amplification.
8.16	After overnight incubation, 500 µl was transferred to 50 ml culture medium for fermentation. Cultures of highly expressed Saccharomyces cerevisiae were carried out and conserved at the end of the four-day culture for subsequent mRNA and resistance assays.
8.19	Obtaining the vector pYET without TPS1 and promoter.
8.21	Amplification of promoter PGK1, TEF2, TDH3 from the dried promoter powder obtained from the order.
8.21	Ligate vector, promoter, TPS gene by ligase. Introduced into receptor E. coli DH5-α and coated plates.
8.22	E. coli labelled with TPS1 gene using PGK1 promoter did not show any colonies in overnight culture, suspecting that the concentration of plasmid and promoter fragments is not enough, re-PCR promoter PGK1,TEF2,TDH3,TPS gene. Gel electrophoresis was carried out and the gel was cut and recovered.
8.23	Connect the vector, promoter and TPS gene again by ligase. Introduced into the sensory E. coli DH5-α and coated plates. No colonies appeared in the overnight culture.
8.24	One day after incubation, colonies appeared on the plate, picked for colony PCR verification, no target band appeared. Recombinant plasmid construction failed.
8.26	After discussion, we concluded that there was a problem with the plate and reformulated the plate with ampicillin solution. And do PCR promoter PGK1, TET2 again, perform gel electrophoresis test, and cut gel recovery experiments.
8.27	Ligate vector, promoter, TPS gene by ligase again. Introduced into the receptor E. coli DH5-α and coated plates. Overnight incubation.
8.28	Tested plate, still no colonies. It is thought that there may be a problem with the ligation. Repeat the ligation. Receptorised E. coli DH5-α was introduced and plates were coated. No colonies appeared in overnight culture.
8.29	Re-ligated the three promoter genes (PGK1,TEF2,TDH3) with TPS1 and plasmid vector separately, colonies appeared in overnight cultures of yeast using the TEF2,TDH3 promoters, and no colonies appeared in yeast using PGK1.
8.30	Ligation of TPS1 and plasmid pYET without removal of promoter ADH2p, introduced into sensory E. coli DH5-α. Colonies appeared in overnight culture.
8.31	Pick E. coli monoclonal clones, make suspension, partially amplify for one day, partially lysed for PCR verification. tEF2, TDH3, two promoter constructs plasmids were successful.
9.1	Use the E. coli suspension amplified for one day, extract the plasmid, and carry out enzyme digestion to verify that the plasmid constructed with three promoters, TEF2, TDH3, and ADH2P, is successful.
9.2	The plasmids (TDH3, TEF2, pYET containing ADH2p, and empty pYET) were introduced into the sensory receptor yeast S288C and coated in tryptophan-deficient YNB medium, respectively.
9.5	After 48 hours of incubation, no colonies appeared in the tryptophan-deficient YNB medium and it was assumed that the YNB had failed, 6.7g/100ml of YNB was reconstituted, and the tryptophan-deficient YNB medium was reconfigured and coated.
9.8	After 4 days of amplification, the tryptophan-deficient flat had intact colonies, half of a single clone was picked for PCR verification, and the other half of the single clone that was successfully verified was transferred to 10 ml of pre-culture medium for amplification.
9.9	After overnight incubation of the pre-culture solution, 500 µl was transferred to 50 ml of culture medium for fermentation. Cultures of highly expressed Saccharomyces cerevisiae were carried out and at the end of the four-day culture were conserved for subsequent mRNA and resistance assays.
9.17	Experiments to compare the growth of different yeast transformants for resistance were carried out using conserved yeast.
9.23	Use the conserved yeast to extract TOTAL RNA and do RT-qPCR to compare mRNA concentrations and compare different promoter strengths.