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Experiment
Contents
Symbiosis Confirmation ~Co-culture Assay~ |
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1. Summary of the experiment |
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To accomplish the symbiosis of the Company cells and Customer cells, we mixed and co-cultured the two cells. Company’s characteristics are C4HSL-Dependent Survival and 3OC12HSL Production (BBa_K1529302), and Company’s characteristics are the opposite from Customer’s.(BBa_K1529797) (If you want to know about these cells in more detail, see the project page. Each cells’ functions are confirmed.) From these characteristics, the symbiosis between the two cells can be established. This experiment was focused on confirmation of the symbiosis and its condition. We constructed the Company cells containing GFP and Customer cells containing RFP. By checking the fluorescence intensity of GFP with flow cytometer, the symbiosis and its condition was detected. |
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Fig. 3-5-1-1. The genetic circuit of Company and Customer |
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2. Results |
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Fig. 3-5-2-1. The growth of the two cells when co-cultured |
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3. Materials and methods |
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3-1 Construction |
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-Strain |
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All the samples were JM2.300 strain |
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-Plasmids |
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| A. Ptet-GFP-Ptet-RhlR (pSB6A1), Prhl(RL)-CmR-LasI (pSB3K3) ...Company | |
| B. Ptet-RFP-Ptet-LuxR (pSB6A1), Plux-CmR-RhlI (pSB3K3) ...Customer | |
| C. Ptet-GFP-Ptet-RhlR (pSB6A1), PlacIq-CmR (pSB3K3) ...Negative control (Company without 3OC12HSL production) | |
| D. Ptet-RFP-Ptet-LuxR (pSB6A1), PlacIq-CmR (pSB3K3) ...Negative control (Customer without C4HSL production) | |
3-2 Assay Protocol |
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| 1. Prepare 2 overnight cultures for each samples A~D in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12 h. | |
| 2. Dilute the overnight cultures to 1 / 100 in fresh LB medium (3 mL) containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) [fresh culture]. | |
| 3. Incubate the fresh cultures in 37°C until the observed OD590 reaches 0.5.If the OD becomes over 0.5, dilute to 0.5 with LB medium. | |
| 4. Add the culture to 3 mL of LB medium as below. | |
| LB medium contains 50 microg / mL ampicillin, 30 microg / mL kanamycin and 100 microg / mL chloramphenicol. | |
| • A 30 microL ＋ B30 microL | |
| • A 30 microL ＋ D 30 microL | |
| • B 30 microL ＋ D 30 microL | |
| 5. Incubate these samples at 37°C for 6 h. (During that time, measure the optical density every one hour.) | |
| 6. Measure the fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company). | |
4. Reference |
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| 1. Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619 | |
