Our notebook
Week 7/12-7/18
We learned basic experimental techniques: preparation of LB amp medium, the transformation of pRS316 plasmid into E. coli, inoculation, and PCR.
Week 7/19-7/25
SLiCE and iVEC to splice the PCR products from the previous week to HBD3 and CYC1
Plasmid extraction of HBD3 and CYC1
Week 7/26-8/1
Yeast experiments were started this week.
Transformation of HBD3 and CYC1 into YPD medium and BMMD medium yeast
Colony PCR on yeast
Week 8/2-8/8
The assay of the effect of HBD3 secretion on the yeast’s growth
HBD3’s antimicrobial ability on bacteria in a halo assay
Week 8/18-22
Transformation of pRS316 into yeast
Week 8/23-8/29
Assay of antimicrobial susceptibility by bacterial liquid absorption method and halo method
Preparation of cell fibers
Assay of Met-responsiveness of Met17 promoter
Week 8/30-9/5
PCR for pRS316+mCherry cloning
pANB1 cloning
pROX1 cloning
SLiCE of pANB1 and pROX1
Colony PCR for pANB1 and pROX1
Plasmid extraction of pANB1 and pROX1
Transformation of pANB1 and pROX1 into yeast
Week 9/6-10
Colony PCR for the yeast transformed with pANB1 or pROX1 in the previous week
SLiCE (Linear Vector Backbone: pRS316+mCherry, Insert DNA: pANB1)
Overlap Extension PCR (OE-PCR) for the sake of connecting three VP16
Colony PCR for the E. coli transformed with pANB1
Defensin assay
Cell fiber preparation
OE-PCR for the sake of connecting three VP16
pANB1 plasmid was extracted from E. coli
Yeast transformation with pANB1
OE-PCR for the sake of connecting three VP16
Defensin assay
SLiCE (Linear Vector Backbone: pRS316, Insert DNA: LasI)
Colony PCR for the yeast transformed with pANB1
Colony PCR for the E. coli transformed with LasI
Week 9/13-17
Operation check of Met17 promotor
Evaluation of oxygen promoter (pANB1)
Defensin assay
OE-PCR for the sake of connecting three VP16
Defensin assay
OE-PCR for the sake of connecting three VP16
OE-PCR for the sake of connecting three VP16
Week 9/20-24
OE-PCR for the sake of connecting three VP16
SLiCE (Linear Vector Backbone: pRS316+mCherry, Insert DNA: pROX1)
Halo assay
Colony PCR for the E. coli transformed with pROX1
OE-PCR for the sake of connecting three VP16
pROX1 plasmid was extracted from E. coli
Yeast transformation with pROX1
Week 9/27-10/1
SLiCE (Linear Vector Backbone: pRS316+mCherry, Insert DNA: pROX1)
Defensin assay
OE-PCR for the sake of connecting three VP16
Colony PCR for the E. coli transformed with pROX1
pROX1 plasmid was extracted from E. coli
Yeast transformation with pROX1
OE-PCR for the sake of connecting three VP16
OE-PCR for the sake of connecting three VP16
Colony PCR for the yeast transformed with pROX1
Cell fiber preparation
Evaluation of oxygen promoter (pROX1)
OE-PCR for the sake of connecting three VP16
Data from team Kyoto
10/8-10/9
10/8
Using Def1 DTT, Def1 no DTT, Def1 ppt, CecA, LL37, NOP1, NOP1v ,and pet11a purified by His tag, we did HPLC.
We also tried the disk diffusion method using Defencine3 from the UTokyo Team.
We used the outstanding bacteria in the vase and Bacillus subtilis.
Add 100μL of bacteria solution to 40mL of standard agar medium
Leave them in the incubator 27℃, 1day
10/9
The antimicrobial peptide from UTokyo Team might not seem to have the antimicrobial activity.
