DNS result not good, PET11a absorbance value higher than others, no trend
*Better solution: water bath stay longer 2.5 hr better;
Professor: suggest use bacteria directly
Steam Distillation
Week 14 (Aug 11-17)
DNS using bacterial culture and use sonification (A40, 1 min 5 sec on 5 sec off, *5 round) to get enzyme extract for tmr
1L:45mL PBS to wash cell pellet get enzyme extract
Cell culture: centrifuge, discard 950 out of 1mL upper liquid, 50 μL cell + 50 μL CMC on 50C incubation for 2 hrs, add DNS and boil, measure
Enzyme crude extract+conc.
1, 3, 5, 6, 7 (3,5 = pectinase, 3 is thermostable) *[1] no in 4C
Apply Centrifuge Conc. Filter: Microcon - 30 (Using 14,000 rcf (xg) for 10 min, 500 μl each and do it twice (1000 μl in total))
*use TBS to wash off the protein
Repeat DNS with bacteria in -80C (1, 6, 7, pET11a)
Centrifuge: 10,000 rpm 2 min
Prepare enzyme and bacteria of 1，3，5
*3,5 need bacteria; 1,6,7,P need enzyme
Steam Distillation
Week 15 (Aug 18-24)
All enzyme and bacterial culture DNS assay (1, 3, 5, 6, 7) (from 4C fridge and -80 fridge stock)
DNS assay with bacteria in -80 fridge stock (1, 3, 5, 6, 7)
Make 1% pectin with CBS
Measure DNS every T minutes (T= 5 10 15 20 25 30 40 50 60 90 120 150 180)
Steam Distillation
Week 16 (Aug 25-31)
Incubated starter culture (from glycerol stock at -80C) in 5 ml LB with 5 μl Ampicillin.
Incubate overnight (16h) in 37C at 250 rpm.
Make IPTG solution (0.5M, 10 ml)
Extract crude enzyme
Store bacterial culture (50 mL each) in -80C fridge
Redo DNS assay across time with 500 rpm when incubating at 50C with substrate
