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)

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.
   ① 3 mL of LB containing Amp, Kan and 3 microL sterile water
   ② 3 mL of LB containing Amp, Kan and 30 microL of 500μM arabinose (final concentration of arabinose is 1 microM)
   ③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 2 microM)
   ④3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 5 microM)
   ※ As for C and D, the suspension were added only in medium ① and ④. 12. Grow the samples at 37 ℃ for 6 hours.
13. Measure OD590 of all the samples every hour.
14. Start preparing the flow cytometer 1 h before the end of incubation.
15. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4℃.
16. Remove the supernatant by using P1000 pipette.
17. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)
18. Dispense all of each suspension into a disposable tube through a cell strainer.
19. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)

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