Team:Peking/Notebook/BXZhao
From 2010.igem.org
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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): | ||
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- | + | ||
===8.1-8.15=== | ===8.1-8.15=== | ||
1. Localization of Lpp-OmpA-MBP-MerR: | 1. Localization of Lpp-OmpA-MBP-MerR: | ||
+ | |||
Western Blotting | Western Blotting | ||
+ | |||
Collaboration with Xin Teng. | Collaboration with Xin Teng. | ||
===8.16-9.5=== | ===8.16-9.5=== | ||
1. Functional Test of Mercury Bioabsorbent: | 1. Functional Test of Mercury Bioabsorbent: | ||
+ | |||
ICP-AES Sample Preparation: | ICP-AES Sample Preparation: | ||
+ | |||
1-1. Grow 10mL E.coli to OD600=0.6 | 1-1. Grow 10mL E.coli to OD600=0.6 | ||
+ | |||
1-2. +1mM IPTG, transfer to 30°C, 30min. | 1-2. +1mM IPTG, transfer to 30°C, 30min. | ||
+ | |||
1-3. +10 uM HgCl2, 30°C overnight expression. | 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. | + | |
+ | 1-4. 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. | 1-5. 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. | 1-6. Freeze-dry the sample, measure the weight of bacteria pellet. | ||
+ | |||
1-7. Resuspend sample with 5mL 2% nitric acid, send for inspection. | 1-7. Resuspend sample with 5mL 2% nitric acid, send for inspection. | ||
+ | |||
+ | ==September== | ||
+ | {| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff" | ||
+ | |- | ||
+ | |style="text-align:center"| Mon | ||
+ | |style="text-align:center"| Tue | ||
+ | |style="text-align:center"| Wed | ||
+ | |style="text-align:center"| Thu | ||
+ | |style="text-align:center"| Fri | ||
+ | |style="text-align:center"| Sat | ||
+ | |style="text-align:center"| Sun | ||
+ | |- | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#8.16-9.5|1]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#8.16-9.5|2]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#8.16-9.5|3]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#8.16-9.5|4]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#8.16-9.5|5]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.6-9.12|6]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#9.6-9.12|7]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#9.6-9.12|8]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.6-9.12|9]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.6-9.12|10]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.6-9.12|11]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.6-9.12|12]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.13-9.17|13]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#9.13-9.17|14]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.13-9.17|15]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.13-9.17|16]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.13-9.17|17]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.18-9.25|18]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.18-9.25|19]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.18-9.25|20]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.18-9.25|21]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#9.18-9.25|22]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.18-9.25|23]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#9.18-9.25|24]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#9.18-9.25|25]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.26-10.2|26]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.26-10.2|27]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#9.26-10.2|28]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.26-10.2|29]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#9.26-10.2|30]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |} | ||
+ | [<html><a href="#top">TOP</a></html>] | ||
+ | ===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== | ||
+ | |||
+ | {| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff" | ||
+ | |- | ||
+ | |style="text-align:center"| Mon | ||
+ | |style="text-align:center"| Tue | ||
+ | |style="text-align:center"| Wed | ||
+ | |style="text-align:center"| Thu | ||
+ | |style="text-align:center"| Fri | ||
+ | |style="text-align:center"| Sat | ||
+ | |style="text-align:center"| Sun | ||
+ | |- | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"|- | ||
+ | |style="text-align:center"|- | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#9.26-10.2|1]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#9.26-10.2|2]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#10.3-10.12|3]] | ||
+ | |- | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#10.3-10.12|4]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#10.3-10.12|5]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#10.3-10.12|6]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#10.3-10.12|7]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#10.3-10.12|8]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#10.3-10.12|9]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#10.3-10.12|10]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#10.3-10.12|11]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#10.3-10.12|12]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#10.13-10.19|13]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#10.13-10.19|14]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#10.13-10.19|15]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#10.13-10.19|16]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#10.13-10.19|17]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#10.13-10.19|18]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#10.13-10.19|19]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#10.20-10.26|20]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#10.20-10.26|21]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#10.20-10.26|22]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/BXZhao#10.20-10.26|23]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#10.20-10.26|24]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#10.20-10.26|25]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/BXZhao#10.20-10.26|26]] | ||
+ | |style="text-align:center"|27 | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |} | ||
+ | [<html><a href="#top">TOP</a></html>] | ||
+ | ===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. | ||
+ | |||
+ | <html> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | <div id="project description bottom"> | ||
+ | <div id="bottomgreen"> | ||
+ | <br> | ||
+ | <a href="https://2010.igem.org/Team:Peking/Team/BXZhao"><font color=#FFFFFF>==go to his page==</font></a> | ||
+ | </div> | ||
+ | </html> |
Latest revision as of 10:23, 27 October 2010
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.
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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.