Team:Peking/Notebook/BXZhao

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===7.4-7.11===
===7.4-7.11===
1. PCR and Subcloning of Lpp-OmpA-MBP-MerR (LOM-MerR):
1. PCR and Subcloning of Lpp-OmpA-MBP-MerR (LOM-MerR):

Revision as of 10:21, 27 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.

September

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 - - -

[TOP]

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.

9.6-9.12

1. Expression and Localization of Lpp-OmpA-MBP-PbrR (LOM-P):

1-1. Transform pET21a-LOM-P into BL21(DE3), plating, overnight culture.

1-2. Pick single colony, amplify culture overnight.

1-3. Secondary amplification, grow until OD600=0.9, add 1mM IPTG, expression overnight.

1-4. Collect bacteria from 1mL medium. Lyse with Bugbuster (Novagen, USA) according to manufacturer’s protocol.

1-5. Perform high speed centrifugation to separate membrane protein with cytosolic protein.

1-6. Add 4×sample buffer, boil at 95°C for 5 min.

1-7. Run SDS-PAGE and western blotting. Verify protein expression and localization.

9.13-9.17

1. Expression of Mercury and Lead Bioabsorbent:

1-1. Transformation, plating, picking and culturing BL21(DE3) expressing LOM-PbrR or LOM-MerR respectively.

1-2. First and secondary amplification, grow to OD600=0.63, add 1mL IPTG, transfer to 30°C, culturing 30min.

1-3. Add different amounts of mercury/lead: 0, 0.1 uM, 1uM, 10uM, respectively. Overnight expression at 30°C.

9.18-9.25

1. Functional Test of Mercury Bioabsorbent LOM-MerR:

1-1. Protein expression according to previous protocol. Amplify bacteria to OD600=0.75, add 1mM IPTG, transfer to 30°C, culturing 30min.

1-2. Divide bacteria into 100mL aliquots, add different amount of mercury. (0, 0.01uM, 0.1uM, 1uM) Overnight expression at 25 °C.

2. ICP-AES Measurement:

2-1. Sample preparation according to previous protocol. Collect bacteria, wash for 4 times, freeze dry overnight.

2-2. Precise weighing bacteria pellet in digestion tube. Resuspend with 8mL fuming nitric acid. Microwave digestion.

2-3. Resuspend sample to 25mL with water. ICP-AES measurement for three parallel times.

9.26-10.2

1. Functional Test of Mercury Bioabsorbent MBP+DsbA-MBP+Lpp-OmpA-MBP-MerR (PML-MerR):

1-1. Protein expression according to previous protocol. Amplify bacteria to OD600=0.5, add 1mM IPTG, transfer to 30°C, culturing 30min.

1-2. Divide bacteria into 100mL aliquots, add different amount of mercury. (0, 0.01uM, 1uM) 24 h expression at 37 °C.

1-3. ICP-AES sample preparation according to previous protocol. Collect bacteria, wash for 4 times, freeze dry overnight. Digest with fuming nitric acid.

October

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 - - - -

[TOP]

9.26-10.2

1. Functional Test of Mercury Bioabsorbent MBP+DsbA-MBP+Lpp-OmpA-MBP-MerR (PML-MerR):

1-1. Protein expression according to previous protocol. Amplify bacteria to OD600=0.5, add 1mM IPTG, transfer to 30°C, culturing 30min.

1-2. Divide bacteria into 100mL aliquots, add different amount of mercury. (0, 0.01uM, 1uM) 24 h expression at 37 °C.

1-3. ICP-AES sample preparation according to previous protocol. Collect bacteria, wash for 4 times, freeze dry overnight. Digest with fuming nitric acid.

10.3-10.12

Full Range Functional Test of Mercury Bioabsorbent (MBP, DsbA-MBP, LOM, PML-MerR):

1. Protein expression according to previous protocol:

1-1. PML-MerR: Amplify bacteria to OD600=0.6, add 1mM IPTG, transfer to 30°C, culturing 30min. Then add different amount of mercury. (0, 0.1uM, 1uM, 10uM) ~40h expression at 30°C.

1-2. MBP, DsbA-MBP, LOM-MerR: Amplify bacteria to OD600=~1, add 1mM IPTG, transfer to 30°C, culturing 30min. Then add 10uM mercury. ~40h expression at 30°C.

1-3. Blank-1: Add 1mM IPTG only. Blank-2: Add 1mM IPTG and 10uM mercury.

2. ICP-AES Measurement:

2-1. Sample preparation according to previous protocol. Collect bacteria, wash for several times, freeze dry overnight.

2-2. Precise weighing bacteria pellet in digestion tube. Resuspend with 8mL fuming nitric acid. Microwave digestion.

2-3. Resuspend sample to 10.00mL with water. ICP-AES measurement for three parallel times.


10.13-10.19

1. Synthesis of Organic Heavy Metal Indicator TritonX-100-PAN-S (TPS):

1-1. Mix 0.2g PAN with 5mL sulfuric acid in a 50mL beaker. Stirring reaction overnight at room temperature.

1-2. Add excessive ethyl ether into reaction mixture, perform suction filtration, wash with acetone and water for several times.

1-3. Collect crude product on the filter paper. Parching overnight on watch glass at 100°C. Collect final product PAN-S.

1-4. Mix 1mg PAN-S with 20mg TritonX-100 and 1mL ddH2O, dissolve thoroughly to get final working solution with orange color. Store final working solution at room temperature.

2. Characterization of Organic Heavy Metal Indicator TritonX-100-PAN-S:

2-1. pH and concentration titration: Add TPS into different pH solution at different mercury concentration to decide proper color transition point. Result shows at pH=7-8, the lower limit of color transition metal concentration is 0.8×10-5M. Color changes from rosy color to bright yellow.

3. Direct Visualization of Mercury Absorbent Function:

3-1. Culturing bacteria with PML-MerR and constitutive promoter overnight.

3-2. Prepare three groups of solution: A: 500uL PBS buffer +10uL TPS +10uM mercury + bacteria pellet (collect from 500uL medium); B: 500uL PBS buffer +10uL TPS +10uM mercury; C: 500uL PBS buffer +10uL TPS.

3-3. 37°C culturing for 1h. After centrifugation, collect upper solution to compare the color change.

3-4. Similar results repeated in HEPES buffer at pH=~8. Pictures taken for view.

10.20-10.26

Full Range Functional Test of Lead Bioabsorbent (MBP, DsbA-MBP, LOM, PML-PbrR):

1. Protein expression according to previous protocol:

1-1. PML-MerR: Amplify bacteria to OD600=0.6, add 1mM IPTG, transfer to 30°C, culturing 30min. Then add different amount of lead. (0, 0.1uM, 1uM, 10uM) ~40h expression at 30°C.

1-2. MBP, DsbA-MBP, LOM-MerR: Amplify bacteria to OD600=~1, add 1mM IPTG, transfer to 30°C, culturing 30min. Then add 10uM lead. ~40h expression at 30°C.

1-3. Blank-1: Add 1mM IPTG only. Blank-2: Add 1mM IPTG and 10uM lead.