Confirm topic: Eczema
FLG, HSP70, fish collagen
Narrow gene of interest to FLG
Lab work within school
Lab work within school and HKU
Initial project proposal(March)
We proposed that combined treatment of these proteins with artificial FLG in coconut oil could restore the expression of FLG while promoting its stability and overall relieving AD symptoms. We emailed our proposal to HKU and CUHK for further advice.
CUHK meeting(8/4)
With their guidance, we narrowed down the three proposed proteins—FLG, HSP70, and fish collagen—to FLG alone, as the other two presented challenges: HSP70 is widely studied in other laboratories, and fish collagen proved too complex for our current capabilities. Additionally, they suggested that a skin model, transwell assay, and scratch assay could serve as pivotal methodologies for our experiments.
HKU meeting(28/4)
This has allowed us to refine our project ideas, including creating disease models in cells by using inflammatory-inducing genes (eg LPS, IL4, IL13) to trigger inflammatory response, instead of our original plan of using FLG knocked out cells, which we found out is not only difficult to execute, but also how it doesn’t really equate to AD cells. Furthermore, they also enriched our knowledge on FLG, NMF and experiment procedures on protein expression and extraction (IPTG induction and SDS Page etc.).
Day Count | Date | Work | Link to protocol | Notes |
Day 1 | 13/5 | Media preparation | ||
Day 2 | 14/5 | Hydration of plasmid | ||
Day 3 | 15/5 | Transformation and plating | ||
Day 4 | 16/5 | Review transformation results | Fail, no colonies observed | |
Day 5 | 19/5 | Transformation | Modification: 45 seconds for heat shock | |
Day 6 | 20/5 | Review transformation results | Success, although one negative control is contaminated | |
Day 6 | 20/5 | Growing single bacteria colony | ||
Day 7 | 21/5 | Plasmid isolation | No.1,3,4 were dropped and spilled | |
Day 7 | 21/5 | Nanodrop | All the sample concentration are very low | |
Day 8 | 26/5 | Inoculation | ||
Day 9 | 27/5 | Plasmid isolation and nanodrop | Highest concentration: 113ng/uL | |
Day 10 | 28/5 | Restriction digestion and gel electrophoresis | ||
Day 11 | 25/6 | Media | ||
Day 12 | 26/6 | Workshop at CUHK | Seeded cells and did scratch assay | |
Day 13 | 27/6 | Workshop at CUHK | ||
Day 14 | 3/7 | Transformation | ||
Day 15 | 10/7 | ColonyPCR preparation | ||
Day 16 | 14/7 | ColonyPCR | ||
Day 17 | 15/7 | Gel electrophoresis and colonyPCR | 15/7 Day17 Gel electrophoresis and single colony inoculation | No bands observed, failed |
Day 18 | 16/7 | Gel electrophoresis | ||
Day 19 | 17/7 | IPTG induction | ||
Day 20 | 18/7 | Cell harvesting | ||
Day 21 | 22/7 | Protein extraction | ||
Day 22 | 23/7 | colonyPCR and cell harvesting | ||
Day 23 | 24/7 | SDS-PAGE | ||
Day 24 | 25/7 | Sample preparation for SDS PAGE for 0.5mM | ||
Day 25 | 28/7 | SDS-PAGE for 0.5mM | ||
Day 26 | 30/7 | Sample preparation for 1mM SDS-PAGE | ||
Day 27 | 31/7 | Destaining of SDS PAGE 1mM and Bacteria growth curve | Dont know how to use the spectrophotometer | |
Day 28 | 1/8 | Bacteria growth curve at37°C | ||
Day 29 | 4/8 | Bacteria growth curve at 37°C | ||
Day 30 | 6/8 | Bacteria growth curve at 37°C | ||
Day 31 | 7/8 | Bacteria growth curve at 37°C and Cell lysate | ||
Day 32 | 8/8 | Bacteria growth curve at 37°C | ||
Day 33 | 11/8 | preparing primary culture, FLG purification at RT, cell lysate | ||
Day 34 | 12/8 | IPTG expression at 20°C, bacterial growth curve, FLG purification at 37°C | ||
Day 35 | 13/8 | IPTG expression at 20°C, preparing FLG at 37°C for SDS-PAGE | ||
Day 36 | 15/8 | |||
Day 37 | 18/8 | SDS-PAGE for total protein at 20°C | ||
Day 38 | 19/8 | Destaining of SDS-PAGE gel | ||
Day 39 | 20/8 | Dry lab work | ||
Day 40 | 21/8 | Dry lab work | ||
Day 41 | 25/8 | HKU lab Seeding cells | ||
Day 42 | 27/8 | HKU lab preparing stock, working concentration and wells | ||
Day 43 | 28/8 | HKU lab harvesting cells at T24 | ||
Day 44 | 29/8 | HKU lab harvesting cells at T48 | ||
Day 45 | 2/9 | HKU lab Extracting RNA T1 | ||
Day 46 | 3/9 | CYCLE 2 START Media preparation 200ml agar, 10 plates | ||
Day 47 | 4/9 | Transformation and plating | ||
Day 48 | 5/9 | HKU lab Extracting RNA T24 and T48 | ||
Day 49 | 9/9 | HKU lab reverse transcription of all samples | ||
Day 50 | 10/9 | Colony PCR for idT-pET-FLG-RFP | ||
Day 51 | 11/9 | Gel electrophoresis | ||
Day 52 | 16/9 | Single colony inoculation | ||
Day 53 | 17/9 | Plasmid isolation + nanodrop | ||
Day 54 | 18/9 | HKU qPCR | ||
Day 55 | 19/9 | RD for confirmation of clone | Failed again ah;( |