Team:Tsinghua/Lab
Part I: Construction of the reporter system
3/17/2017 By HJN
Transform the pRS-HXT2 and pRS-HXT5 plasmid (Leu) to amplify them.
3/19/2017 By HJN&ZSY
Pick the pRS-HXT2 and pRS-HXT5 clones. Shake in LB+Carb medium for 10hrs. Extract the plasmid.
3/21/2017 By HJN&ZSY
Restriction enzyme digestion to validate the pRS-HXT2 and pRS-HXT5 plasmid.
3/23/2017 By HJN&ZSY
Culture the HXT-auxotroph yeast in YPM medium.
3/26/2017 By HJN&ZSY
Transform pRS-HXT2 or pRS-HXT5 or null into HXT-auxotroph yeast (named HXT2 and HXT5 yeast, respectively). Culture the former two in the Sc-Leu medium and the latter one in YPM medium.
3/30/2017 By HJN&ZSY
Serial dilution test of the HXT2 and HXT5 yeast as well as the control on the YPD and Sc-Leu medium, two replicas each.
4/7/2017 By HJN
PCR the target segment from pYMN6-KanMX plasmid for the KO of Gal4 gene in the HXT2/5 yeast strain.
4/8/2017 By HJN
Transform the pYMN
6-KanMX PCR product into the HXT-auxotroph yeast (named EBYWV4000-GAL4Δ(GAL80) ).
4/12/2017 By HJN
Extract the genomic DNA of EBYWV4000-GAL4Δ(GAL80).
4/13/2017 By HJN
PCR detection of EBYWV4000-GAL4Δ(GAL80) genome.
4/20/2017 By HJN
PCR pYM17(Net) with the GAL80 homolog. Transform it into the EBYWV4000-GAL4Δ(GAL80) to get the JDYY001 yeast strain.
5/18/2017 By LDY&MHP
PCR the pGALS and clean it up. Cut it and the HXT2 plasmid with DraIII and XhoI
5/19/2017 By HJN
Gel extraction of pGALS. Ligation and transformation.
5/20/2017 By HJN
Clony PCR of pGAL-HXT2. Culture the correct one with LB+Carb.
5/21/2017 By HJN
Extract the pGAL-HXT2, validate it by XbaI and send sequencing.
5/28/2017 By ZSY
Transform pGAL-HXT2 and pPC86-AD-BD to JDYY001 and culture it on Sc-Leu-Trp.
6/19/2017 By GXF
Yeast transformation of pGAL-HXT&AD-BD or pGAL-HXT or HXT to JDYY001
6/21/2017 By HY
PCR URA3.
By LYX
Clean URA3 up.
6/22/2017 By HY
Use AftII to cut the URA3 segment and pGAL-HXT2. Dephosphorylate the vector with CIP. Ligate them.
6/23/2017 By YTR
Transform the Ligated URA3-pGAL-HXT2.
6/27-7/6/2017 By HY&YTR
Reconstruct the URA3-pGAL-HXT2.
8/9-13 By HJN
Reconstruct the URA3-pGAL-HXT2. This time we succeed.
Part II: Construction of AD/BD-ScFv1/2 detector
4/1/2017 By HJN
Transform pDBLeu-BD, pPC86-AD and the 6-11K plasmid in the distribution plate.
4/2/2017 By HJN
Pick the pDBLeu-BD, pPC86-AD and the 6-11K clones. Extract the plasmid. Restriction enzyme digestion to validate the pDBLeu-BD and pPC86-AD plasmid.
4/5/2017 By HJN
PCR of BD by primer 43/44, PCR clean-up and cut it, as well as the pPC86-AD plasmid by XmaI and EcoRI.
4/6/2017 By HJN
Gel extraction of cut BD and pPC86-AD. Ligate them and transform in the DH5α (named pPC86-AD-BD).
4/7/2017 By HJN
Clony PCR of pPC86-AD-BD. Culture the correct ones.
4/8/2017 By HJN
Extract the pPC86-AD-BD plasmid. Validate it by BsaI and send it to sequencing.
4/12/2017 By HJN
Extract the ScFv1 and ScFv2 plasmids synthesized by Wuxi Qinglan Biotech Co. Ltd. Validate it by PstI and XmaI.
4/15/2017 By HJN&ZSY
PCR the ScFv1, ScFv1-His, ScFv2, ScFv2-His and clean it up. By YTR
PCR the AD, AD-His, BD, BD-His and clean it up
4/16/2017 By HJN&ZSY
Cut the ScFv1, ScFv1-His, ScFv2, ScFv2-His segments as well as the pPC86-AD/BD plasmid by XmaI and SpeI. Do gel extraction and PCR clean-up. Ligate and transform them (named AD-ScFv1, AD-ScFv1-His, BD-ScFv2, BD-ScFv2-His plasmid). By YTR
POT assembly[1] of ScFv1-AD, ScFv1-AD-His, ScFv2-BD, ScFv2-BD-His plasmid and transform them.
4/20/2017 By HJN&ZSY
Extract the plasmid of AD-ScFv1, AD-ScFv1-His, BD-ScFv2, BD-ScFv2-His, ScFv1-AD, ScFv1-AD-His, ScFv2-BD, ScFv2-BD-His. Validate the former four by PstI and BamHI and send sequencing. By YTR
Validate ScFv1-AD, ScFv1-AD-His, ScFv2-BD, ScFv2-BD-His plasmid by SacI. Send correct ScFv1-AD and ScFv2-BD-His to sequence them.
4/23-24/2017 By YTR
Still trying to find the correct ScFv2-BD and ScFv1-AD-His.
4/25/2017 By YTR
POT assembly[1] of ScFv2-BD again.
4/26/2017 By ZSY
Transform AD-ScFv1& BD-ScFv2-His or AD-ScFv1-His& BD-ScFv2 or pPC86-AD-BD to JDY26 yeast. Culture the former two on Sc-Trp-Leu while the latter one on Sc-Trp.
4/27/2017 By LDY&MHP
Transform the new assembled ScFv2-BD plasmid. By ZSY
Transform the JDY26 with AD-ScFv1& BD-ScFv2 or ScFv1-AD& BD-ScFv2
5/5/2017 By YTR
Extract the ScFv2-BD plasmid and validate it with SacI.
8/7/2017 By ZSY
Cut the AD-ScFv1 with SalI and dephosphorylate it with CIP. Do gel extraction. Anneal the 3*flag insert. Ligate the two (named AD-3f-ScFv1).
8/8/2017 By ZSY
Transform the ligated AD-3f-ScFv1.
8/9/2017 By ZSY
Clony PCR of AD-3f-ScFv1. Culture the correct one in LB+Carb.
8/10/2017 By ZSY
Extract AD-3f-ScFv1 and validate it by ClaI and XbaI. Send sequencing.
Part III: Combination exploration of the system.
6/22/2017 By YTR
Get the function curve of the glucometer in culture medium Sc-Ura-Trp.
7/7/2017 By LDY
Yeast transformation AD-ScFv1, BD-ScFv2 and URA3-pGAL-HXT2.
7/28/2017 By LDY
Yeast transformation AD-ScFv1, BD-ScFv2 and URA3-pGAL-HXT2.
8/17/2017 By HJN
Yeast transformation AD-ScFv1, BD-ScFv2&URA3-pGAL-HXT2 or AD-ScFv1&BD-ScFv2 or null.
8/22/2017 By LDY
Get the function curve of glucometer using both water and culture medium (Sc-maltose) as solution.
9/9&13/2017 By HY
Transfect constitutively expressed HXT2 into JDYY001.
9/13-15/2017 By LDY
POT assembly[1] of pGALS-GFP
9/23/2017 By ZSY
Reconstruct the BD-ScFv by PCR the plasmid.
9/24/2017 By HJN&LYX
Extract the PCR product the day before. Ligate and transform it.
9/25/2017 By HY
Use JDYY001 stably expressing HXT to see how it consume glucose.
9/28/2017 By JZR
Use JDYY001 transfected with pGALS-GFP and AD-ScFv1, BD-ScFv1 cultured in AFT O/N to make slides.
By GBY
Use confocal to take the photo of the yeast in 488nm, 100* oil microscope.
10/2-10/2017 By ZSY
Transform the yeast with pGAL-HXT together with AD-BD and see whether it can grow on YPD medium, which use glucose as its only carbon source.
10/13-23/2017 By LYX&HJN&ZSY&HY&LDY&GBY
Parts assembly
10/19-23/2017 By GBY
Clone pGALS-GFP on 2u plasmid
Reference
1. Guo, Y., et al., YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res, 2015. 43(13): p. e88.
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