contents

1. Introduction

2. Summary of the Experiment

3. Results

3.1. Arabinose dependent FimE expression

3.2. FLA analysis

4. Materials and Methods

4.1. Construction

4.2. Assay Protocol

4.2.1 Arabinose dependent FimE expression

4.2.2. FLA analysis

6. Reference

1. Introduction

　　　　　　

To confirm the function of fim switch in the presence of FimE, we constructed two new genetic circuit parts, BBa_K1632013 and BBa_K1632002 (Fig. 3-5-1). BBa_K1632013 enables arabinose-inducible expression of wild type FimE. In BBa_K1632013 and BBa_K1632002, either [ON] or [OFF] fim switch is placed upstream of GFP coding sequence.

Fig.3-7-2-1.  New plasmids we constructed to confirm the function of fim switch

2. Summary of the Experiment

　　　　　　

Our purpose is to confirm that FimE inverts fimswitch only from ON to OFF. Taking endogenous FimB and FimE into account, we prepared six plasmids sets shown below. We measured the fluorescence intensity by GFP expression when we added arabinose.　また、我々はFimSが本当に反転しているかどうかを確認するために、FLAを使った解析とシークエンスデータの解析を行いました。

Fig.3-7-2-1. Plasmids for the experiment of FimE dependent fim switch state assay

3. Results

3.1. Arabinose dependent FimE expression

　　　　　　

Fig.3-5-3-1 はフローサイトメーターで測定した各サンプルのヒストグラムである。レポーターセルCとDの結果から、内在性のFImBとFimEは無視できることがわかる。また、レポーターセルE,Fの結果から、FImEの発現はヒストグラムの波形にほとんど影響を与えないことがわかる。 　レポーターセルAの結果は、FimEの発現量が増加すると、蛍光強度が減少することを示している。このことより、我々はFimSがFimEによってOn状態からOff状態に切り替わっていることを確信した。また、レポーターセルBの結果はFimEの発現量が増加してもレポーターセルBのGFP発現量は変化しないことを示している。これより、fimSはFimEによってOff状態からOn状態に切り替わらないことを確信した。以上二つのレポーターセルA,Bの結果より、FimEはFimSをinverts fimswitch only from ON to OFF していることを確信した。

Fig. 3-5-3-1.

3.2. FLA analysis

　　　　　　

写真とシークエンスデータ

4. Materials and Methods

4.1. 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. promoter less M256ICysE(pSB6A1)+ fim switch[default ON](wild-type)_gfp(pSB3K3)…Positive control 1

Fig. 3-5-4-3.

　　　　　　

D. promoter less M256ICysE(pSB6A1)+ fim switch[default OFF](wild-type)_gfp(pSB3K3)…Negative control 1

Fig. 3-5-4-4.

　　　　　　

E. Pbad/araC-fimE (pSB6A1) +J23119 promoter_gfp (pSB3K3)…Positive control2

Fig. 3-5-4-5.

　　　　　　

F. Pbad/araC-fimE (pSB6A1) +promoter less gfp (pSB3K3)…Negative control2

Fig. 3-5-4-6.

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. 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. Add 1 mL of LB containing Amp and Kan, and suspend.
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

1. After the assay of “Arabinose dependent FimB expression”, miniprep cell culture (A,B, ,C and D) of leftover as here.(http://parts.igem.org/Help:Protocols/Miniprep)
2. Turn on water bath to 42℃.
3. Take competent DH5alpha strain from -80℃ freezer and leave at rest on ice.
4. Add 3 µl of each plasmids in a 1.5 ml tube.
5. Put 25 µl competent cell into each 1.5 ml tubes with plasmid.
6. Incubate on ice for 15 min.
7. Put tubes with DNA and competent cells into water bath at 42℃ for 30 seconds.
8. Put tubes back on ice for 2 minutes.
9. Add 125 µl of SOC medium. Incubate tubes for 30 minutes at 37℃.
10. Make a 1:5 dilution in 150µl of fresh SOC medium.
11. Spread about 100 µl of the resulting culture of LB plate containing kanamycin.
12. Incubate LB plate for 14-15 hours at 37℃.

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