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</br> | </br> | ||
<h2>1. Construction of the library of BL21 (DE3)-derived variant strains</h2> | <h2>1. Construction of the library of BL21 (DE3)-derived variant strains</h2> | ||
− | < | + | <h5><b>Materials & Apparatus</b></h5> |
<h6>1. LB medium with 50 μg/mL kanamycin </h6> | <h6>1. LB medium with 50 μg/mL kanamycin </h6> | ||
<h6>2. LB medium with 50 μg/mL spectinomycin</h6> | <h6>2. LB medium with 50 μg/mL spectinomycin</h6> | ||
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</br> | </br> | ||
<h2>1.1 CRISPR</h2> | <h2>1.1 CRISPR</h2> | ||
− | < | + | <h5><b>Before Experiment</b></h5> |
<h6>1. 10μL bacteria solution was inoculated into 5mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.</h6> | <h6>1. 10μL bacteria solution was inoculated into 5mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.</h6> | ||
<h6>2. Pellet 1.5mL bacteria solution from step 1 in a clean 2mL microcentrifuge tube by centrifugation at 4,000 rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.</h6> | <h6>2. Pellet 1.5mL bacteria solution from step 1 in a clean 2mL microcentrifuge tube by centrifugation at 4,000 rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.</h6> | ||
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<h6>9. Select the single bacteria on the LB solid plate, insert it into the LB liquid medium and cultivate for at least 12-16h.</h6> | <h6>9. Select the single bacteria on the LB solid plate, insert it into the LB liquid medium and cultivate for at least 12-16h.</h6> | ||
</br> | </br> | ||
− | < | + | <h5><b>Experiment</b></h5> |
<h6>1. 10μL bacteria which contains pCas plasmid solution and 5μL kanamycin were inoculated into 5mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.</h6> | <h6>1. 10μL bacteria which contains pCas plasmid solution and 5μL kanamycin were inoculated into 5mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.</h6> | ||
<h6>2. Pellet 1.5mL bacteria solution from step 1 in a clean 2mL microcentrifuge tube by centrifugation at 4,000 rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.</h6> | <h6>2. Pellet 1.5mL bacteria solution from step 1 in a clean 2mL microcentrifuge tube by centrifugation at 4,000 rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.</h6> | ||
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</br> | </br> | ||
<h2>1.2 CBE</h2> | <h2>1.2 CBE</h2> | ||
− | < | + | <h5><b>Before Experiment</b></h5> |
<h6>1. Use CRISPR editing technology to replace the RBS sequence on the BL21(DE3) genome with GGGGGGGG, named BL21(DE3)-8G.</h6> | <h6>1. Use CRISPR editing technology to replace the RBS sequence on the BL21(DE3) genome with GGGGGGGG, named BL21(DE3)-8G.</h6> | ||
<h6>2. 10μL BL21 (DE3)-8G bacteria solution was inoculated into 5mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.</h6> | <h6>2. 10μL BL21 (DE3)-8G bacteria solution was inoculated into 5mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.</h6> | ||
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</br> | </br> | ||
− | < | + | <h5><b>Experiment</b></h5> |
<h6>1. 10μL bacteria which contains pdCas-CDA-UGI-LVA plasmid solution and 5μL kanamycin were inoculated into 5mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.</h6> | <h6>1. 10μL bacteria which contains pdCas-CDA-UGI-LVA plasmid solution and 5μL kanamycin were inoculated into 5mL LB liquid mediums at 37 °C, 200 rpm and last 12-16h.</h6> | ||
<h6>2. Pellet 1.5mL bacteria solution from step 1 in a clean 2mL microcentrifuge tube by centrifugation at 4,000 rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.</h6> | <h6>2. Pellet 1.5mL bacteria solution from step 1 in a clean 2mL microcentrifuge tube by centrifugation at 4,000 rpm for 10 minute at 4℃ temperature. Decant or aspirate medium and discard.</h6> | ||
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</br> | </br> | ||
<h2>2. Fluorescence intensity measurement to screening high-yield strains</h2> | <h2>2. Fluorescence intensity measurement to screening high-yield strains</h2> | ||
− | < | + | <h5><b>Material&Apparatus</b></h5> |
<h6>1. Luria Bertani (LB) medium with 50 μg/mL kanamycin (Kan) </h6> | <h6>1. Luria Bertani (LB) medium with 50 μg/mL kanamycin (Kan) </h6> | ||
<h6>2. Terrific Broth (TB) medium with 50 μg/mL Kan</h6> | <h6>2. Terrific Broth (TB) medium with 50 μg/mL Kan</h6> | ||
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<h6>6. Enzyme-labeled instrument</h6> | <h6>6. Enzyme-labeled instrument</h6> | ||
</br> | </br> | ||
− | < | + | <h5><b>Before Experiment</b></h5> |
<h6>1. Single colonies and 0.8μL kanamycin were added into 800μL LB liquid mediums at 37 °C, 1200 rpm and last 12h in the 96-well transparent microplate. </h6> | <h6>1. Single colonies and 0.8μL kanamycin were added into 800μL LB liquid mediums at 37 °C, 1200 rpm and last 12h in the 96-well transparent microplate. </h6> | ||
</br> | </br> | ||
− | < | + | <h5><b>Experiment</b></h5> |
<h6>1. 10μL bacteria solution and 0.8μL kanamycin were added into 800μL TB liquid mediums at 37 °C, 1200 rpm and last 3h in the 96-well transparent microplate. </h6> | <h6>1. 10μL bacteria solution and 0.8μL kanamycin were added into 800μL TB liquid mediums at 37 °C, 1200 rpm and last 3h in the 96-well transparent microplate. </h6> | ||
<h6>2. 10μL IPTG (0.1M) was added into above TB liquid mediums and continued to shake at 28°C, 1200 rpm and last 24h.</h6> | <h6>2. 10μL IPTG (0.1M) was added into above TB liquid mediums and continued to shake at 28°C, 1200 rpm and last 24h.</h6> | ||
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</br> | </br> | ||
<h2>3. Fluorescence intensity measurement of fermentation broth</h2> | <h2>3. Fluorescence intensity measurement of fermentation broth</h2> | ||
− | < | + | <h5><b>Material&Apparatus</b></h5> |
<h6>1. Luria Bertani (LB) medium with 50 μg/mL kanamycin (Kan) </h6> | <h6>1. Luria Bertani (LB) medium with 50 μg/mL kanamycin (Kan) </h6> | ||
<h6>2. Terrific Broth (TB) medium with 50 μg/mL Kan</h6> | <h6>2. Terrific Broth (TB) medium with 50 μg/mL Kan</h6> | ||
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<h6>7. Enzyme-labeled instrument</h6> | <h6>7. Enzyme-labeled instrument</h6> | ||
</br> | </br> | ||
− | < | + | <h5><b>Before Experiment</b></h5> |
<h6>1. Single colonies and 5μL kanamycin were added into 5mL LB liquid mediums at 37 °C, 220 rpm and last 12-16h.</h6> | <h6>1. Single colonies and 5μL kanamycin were added into 5mL LB liquid mediums at 37 °C, 220 rpm and last 12-16h.</h6> | ||
</br> | </br> | ||
− | < | + | <h5><b>Experiment</b></h5> |
<h6>1. 200μL bacteria solution and 30μL kanamycin were added into 30mL TB liquid mediums at 37 °C, 220 rpm and last 3h.</h6> | <h6>1. 200μL bacteria solution and 30μL kanamycin were added into 30mL TB liquid mediums at 37 °C, 220 rpm and last 3h.</h6> | ||
<h6>2. 9μL IPTG(1M) was added into above TB liquid mediums and continued to shake at 28°C, 220 rpm and last 24h.</h6> | <h6>2. 9μL IPTG(1M) was added into above TB liquid mediums and continued to shake at 28°C, 220 rpm and last 24h.</h6> | ||
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</br> | </br> | ||
<h2>4. SDS-PAGE </h2> | <h2>4. SDS-PAGE </h2> | ||
− | < | + | <h5><b>Material&Apparatus</b></h5> |
<h6>1. Luria Bertani (LB) medium with 50 μg/mL kanamycin (Kan) </h6> | <h6>1. Luria Bertani (LB) medium with 50 μg/mL kanamycin (Kan) </h6> | ||
<h6>2. Terrific Broth (TB) medium with 50 μg/mL Kan</h6> | <h6>2. Terrific Broth (TB) medium with 50 μg/mL Kan</h6> | ||
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<h6>12. TEMED</h6> | <h6>12. TEMED</h6> | ||
<h6>13. SDS-PAGE Sample Loading Buffer,10X (produced by Sangon Biotech®)</h6> | <h6>13. SDS-PAGE Sample Loading Buffer,10X (produced by Sangon Biotech®)</h6> | ||
− | <h6>14.Glacial acetic acid</h6> | + | <h6>14. Glacial acetic acid</h6> |
<h6>15. Protein samples</h6> | <h6>15. Protein samples</h6> | ||
<h6>16. Protein marker 180kDa (produced by Sangon Biotech®)</h6> | <h6>16. Protein marker 180kDa (produced by Sangon Biotech®)</h6> | ||
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<h6>19. Microwave oven</h6> | <h6>19. Microwave oven</h6> | ||
</br> | </br> | ||
− | < | + | <h5><b>Before Experiment</b></h5> |
<h6>1. Single colonies and 5μL kanamycin were added into 5mL LB liquid mediums at 37 °C, 220 rpm and last 12-16h.</h6> | <h6>1. Single colonies and 5μL kanamycin were added into 5mL LB liquid mediums at 37 °C, 220 rpm and last 12-16h.</h6> | ||
</br> | </br> | ||
− | < | + | <h5><b>Experiment</b></h5> |
<h6>1. 200μL bacteria solution and 30μL kanamycin were added into 30mL TB liquid mediums at 37 °C, 220 rpm and last 3h.</h6> | <h6>1. 200μL bacteria solution and 30μL kanamycin were added into 30mL TB liquid mediums at 37 °C, 220 rpm and last 3h.</h6> | ||
<h6>2. 9μL IPTG was added into above TB liquid mediums and continued to shake at 28°C, 220 rpm and last 24h.</h6> | <h6>2. 9μL IPTG was added into above TB liquid mediums and continued to shake at 28°C, 220 rpm and last 24h.</h6> | ||
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</br> | </br> | ||
<h2>5. Antibacterial experiment</h2> | <h2>5. Antibacterial experiment</h2> | ||
− | < | + | <h5><b>Material&Apparatus</b></h5> |
<h6>1. Luria Bertani (LB) medium </h6> | <h6>1. Luria Bertani (LB) medium </h6> | ||
<h6>2. Escherichia coli MG1655</h6> | <h6>2. Escherichia coli MG1655</h6> | ||
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<h6>9. 37℃ incubator</h6> | <h6>9. 37℃ incubator</h6> | ||
</br> | </br> | ||
− | < | + | <h5><b>Before Experiment</b></h5> |
<h6>1. Single colony was added into 5mL LB liquid mediums at 37 °C, 220 rpm and last 12-16h.</h6> | <h6>1. Single colony was added into 5mL LB liquid mediums at 37 °C, 220 rpm and last 12-16h.</h6> | ||
</br> | </br> | ||
− | < | + | <h5><b>Experiment</b></h5> |
<h6>1. The 10mL water agar medium were poured into the plates and condensed. | <h6>1. The 10mL water agar medium were poured into the plates and condensed. | ||
</h6> | </h6> | ||
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<h6>4. After 12 hours, the diameter of bacteriostatic zones were measured to evaluate the antibacterial activity of AMPs. | <h6>4. After 12 hours, the diameter of bacteriostatic zones were measured to evaluate the antibacterial activity of AMPs. | ||
</h6> | </h6> | ||
+ | </br> | ||
+ | </br> | ||
</br> | </br> | ||
Revision as of 13:29, 20 October 2021