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

　　　　　　

ここにコピペ。

2. Summary of the Experiment

　　　　　　

ここにコピペ。

3. Results

　　　　　　

ここにコピペ。

4. Discussion

　　　　　　

ここにコピペ。

5. Materials and Methods

5.1. Construction

-Strain

　　　　　　

All the samples were DH5alpha strain.

-Plasmids

　　　　　　

A. Pbad/araC_fimE(wild-type) (pSB6A1)+ fim switch[default ON](wild-type)_gfp (pSB3K3)

Fig.3-5-4-1.

　　　　　　

B. Pbad/araC_fimE(wild-type) (pSB6A1)+ fim switch[default OFF](wild-type)_gfp (pSB3K3)

Fig.3-5-4-2.

　　　　　　

C. rbs_M256ICysE(pSB6A1)+ fim switch[default ON](wild-type)_gfp(pSB3K3)…Positive control 1

Fig.3-5-4-3.

　　　　　　

C. rbs_M256ICysE(pSB6A1)+ fim switch[default OFF](wild-type)_gfp(pSB3K3)…Negative control 1

Fig.3-5-4-4.

　　　　　　

C. rbs_M256ICysE(pSB6A1)+ fim switch[default OFF](wild-type)_gfp(pSB3K3)…Negative control 1

Fig.3-5-4-5.

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