Team:Peking/Notebook/BXZhao

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===8.1-8.15===
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1. Localization of Lpp-OmpA-MBP-MerR:
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Western Blotting
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Collaboration with Xin Teng.
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===8.16-9.5===
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1. Functional Test of Mercury Bioabsorbent:
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ICP-AES Sample Preparation:
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1-1. Grow 10mL E.coli to OD600=0.6
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1-2. +1mM IPTG, transfer to 30°C, 30min.
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1-3. +10 uM HgCl2, 30°C overnight expression.
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1-4. Centrifuge and collect 10mL bacteria at 12000rpm, discard the medium, wash the pellet with ddH2O for a few times, collect by centrifugation.
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1-5. Add 3 mL fuming nitric acid, heat at 65°C for 4h. Wait till NO2 complete release.
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1-6. Freeze-dry the sample, measure the weight of bacteria pellet.
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1-7. Resuspend sample with 5mL 2% nitric acid, send for inspection.

Revision as of 06:06, 26 October 2010





   Boxuan Zhao's Notes
                                                                                                                                                goto his page
My work is merging OmpA and anchor protein Lpp with metal binding domain of MerR to accomplish the goal of surface display. Additionally, I participate the functional testing of whole-cell bioabsorbents. By recognizing the homologous regions among them, I designed the metal binding domain complex for four different MerR family proteins, which can be used to verify the reasonableness of our project design.

download his notes

Contents

July

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]

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.

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