Team:Tokyo Tech/Experiment/FimE dependent fim switch state assay
C4HSL-dependent growth assay
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contents
1. Introduction
2. Summary of the Experiment
3. Results
4. Discussion
5. Materials and Methods
5.1. Construction
5.2. Assay Protocol
5.2.1. Protocol1
5.2.2. Protocol2
6. Reference
1. Introduction
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2. Summary of the Experiment
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3. Results
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4. Discussion
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5. Materials and Methods
5.1. Construction
-Strain
All the samples were DH5alpha strain.
-Plasmids
Device 1: J23101 + I13504(pSB1C3)
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Fig.3-7-4-1. |
4.2. Assay Protocol
4.2.1. Arabinose dependent FimE expression
1. Prepare overnight cultures for the each sample in 3 ml LB medium, containing ampicillin (50 microg / mL), kanamycin (30 microg / mL) and glucose (final concentration of mass of glucose is 1.0 percent) at 37 ℃ for 12h.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration of mass of glucose is 1.0 percent).
3. Make a 1:100 dilution in 3 mL of fresh LB containing antibiotic, and grow the cells at 37 ℃ until the observed OD590 reaches 0.4 (Fresh Culture)
4. After incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25 ℃.
5. Remove the supernatant by using P1000 pipette.
6. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25 ℃
7. Remove the supernatant by using P1000 pipette.
8. Take the samples, and centrifuge at 5000x g, 1 min, 25 ℃.
9. Remove the supernatant by using P1000 pipette.
10. Suspend the pellet in 1 mL of LB containing Amp and Kan.
11. Add 30 microL of suspension in the following medium.
4.2.2. FLA analysis
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5. Reference
1. Timothy S. Ham et al. (2006) A Tightly Regulated Inducible Expression System Utilizing the fim Inversion Recombination Switch. Biotechnol Bioeng 94(1):1-4