Team:Peking/Notebook/BXZhao
From 2010.igem.org
Contents
July
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4 | 5 | 6 | 7 | 8 | 9 | 10 |
11 | 12 | 13 | 14 | 15 | 16 | 17 |
18 | 19 | 20 | 21 | 22 | 23 | 24 |
25 | 26 | 27 | 28 | 29 | 30 | 31 |
[TOP]
7.4-7.11
1. PCR and Subcloning of Lpp-OmpA-MBP-MerR (LOM-MerR):
Collaboration with Junyi Jiao.
2. Promoter Analysis and Alignment of Other Proteins in Family
2-1. Search NCBI for wild type promoter for PbrR, CueR and CupR respectively.
2-2. Amino acid sequence alignment of proteins for similarity analysis.
7.12-7.18
1. Subcloning of LOM-MerR into commercial plasmid (with His-tag for localization):
Collaboration with Junyi Jiao.
2. Structure Prediction and Design of Lead Bioabsorbent:
2-1. Structure prediction and domain functional analysis of PbrR based on alignment with MerR.
2-2. MBP-PbrR design and primer design for the construction of PbrR.
7.19-7.25
1. LOM-MerR Expression:
1-1. Transform pET21a/pSB1a3-LOM-MerR into BL21(DE3), plating, overnight culture.
2-2. Pick single colony, amplify culture overnight.
2-3. Secondary amplification, grow until OD600=~0.6, add 1mM IPTG, expression 5h at 30℃.
2-4. Run SDS-PAGE, verify protein expression.
7.26-7.31
1. LOM-MerR Expression Optimization:
1-1. Transform pET21a/pSB1a3-LOM-MerR into BL21(DE3), plating, overnight culture.
2-2. Pick single colony, amplify culture overnight.
2-3. Secondary amplification, grow until OD600=~0.6, add 1mM IPTG, expression 24h at 16℃.
2-4. Run SDS-PAGE, verify protein expression.
2. Functional Test of Mercury Bioabsorbent:
Dithizone Assay:
Collaboration with Junyi Jiao.
August
Mon | Tue | Wed | Thu | Fri | Sat | Sun |
1 | 2 | 3 | 4 | 5 | 6 | 7 |
8 | 9 | 10 | 11 | 12 | 13 | 14 |
15 | 16 | 17 | 18 | 19 | 20 | 21 |
22 | 23 | 24 | 25 | 26 | 27 | 28 |
29 | 30 | 31 | - | - | - | - |
[TOP]
8.1-8.15
1. Localization of Lpp-OmpA-MBP-MerR:
Western Blotting
Collaboration with Xin Teng.
8.16-9.5
1. Functional Test of Mercury Bioabsorbent:
ICP-AES Sample Preparation:
1-1. Grow 10mL E.coli to OD600=0.6
1-2. +1mM IPTG, transfer to 30°C, 30min.
1-3. +10 uM HgCl2, 30°C overnight expression.
1-4. Centrifuge and collect 10mL bacteria at 12000rpm, discard the medium, wash the pellet with ddH2O for a few times, collect by centrifugation.
1-5. Add 3 mL fuming nitric acid, heat at 65°C for 4h. Wait till NO2 complete release.
1-6. Freeze-dry the sample, measure the weight of bacteria pellet.
1-7. Resuspend sample with 5mL 2% nitric acid, send for inspection.