Team:Peking/Notebook/BXZhao

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Contents

• July, 2010

• August, 2010

• September, 2010

• October, 2010

July

[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

[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.