contents

1. Introduction

2. Summary of the Experiment

3. Results

3.1. Arabinose-dependent FimB (wild-type) expression

3.2. FLA analysis

4. Discussion

5. Materials and Methods

5.1. Construction

5.2. Assay Protocol

5.2.1 Arabinose dependent FimB expression

5.2.2. FLA analysis

6. Reference

1. Introduction

　　　　　　

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

　　　　　　

Fig.3-5-1-1. New plasmids we constructed to confirm the function of fim switch

2. Summary of the Experiment

　　　　　　

Our purpose is to confirm that FimE (wild-type) inverts the fim switch (wild-type) from ON to the OFF and from OFF to ON (Fig.3-5-2-1). We prepared six plasmids below. (Fig.3-5-2-2). We measured the fluorescence intensity from the GFP expression in the presence of arabinose. From the results, we confirmed that our fim switch (wild-type) is inverted from ON to OFF and OFF to ON. From the results we also confirmed our fim switch (wild-type) is not inverted by the endogenous FimB and FimE and that FImE(wild-type) expression doesn’t have effect on the GFP expression. We also confirmed the inversion of our fim switch (wild-type) by コロニーカウンティング以下は篠原よろしく

(1) P_BAD/araC_fimE (pSB6A1)+ fim switch[default ON](wild-type)_GFP (pSB3K3)
(2) P_BAD/araC_fimE (pSB6A1) + fim switch[default OFF](wild-type) _GFP (pSB3K3)
(3)Positive control 1: (pSB6A1)+ fim switch[default ON](wild-type) _GFP (pSB3K3)
(4)Negative control 1: (pSB6A1)+ fim switch[default OFF](wild-type) _GFP (pSB3K3)
(5)Positive control 2: P_BAD/araC_fimE (wild-type) (pSB6A1)+Pcon_GFP (pSB3K3)
(6)Negative control 2: P_BAD/araC_fimE (wild-type) (pSB6A1)+promoter less_GFP (pSB3K3)

Fig.3-5-2-1. Plasmids for the experiment of FimE dependent fim switch state assay

3. Results

3.1. Arabinose dependent FimE(wild-type) expression

　　　　　　

We tried to confirm that fim switch is unidirectionally inverted in the presence of FimE (wild-type) by using GFP as a reporter, under 4 different concentrations of arabinose. In the medium with 0 M arabinose, we supplemented the medium with 1.0 % glucose in order to repress the leakage in the P_BAD/araC promoter. Fig. 3-6-3-1 shows the histograms of the samples measured by the flow cytometer. In the results of the reporter cell (1), when the Induction of FimE(wild-type) expression increases, the fluorescence intensity decreases. From this fact, we confirmed that the fim switch (wild-type) is inverted from ON to OFF by FimE (wild-type). From the result of the reporter cell (2), even when the expression amount of FimE (wild-type) increases, the expression amount of GFP in the reporter cell (2) does not change. From this fact, we confirmed that the fim switch (wild-type) is inverted only from ON to OFF by FimE (wild-type). From the results of the two reporter cells (1) and (2), we successfully confirmed that FimE (wild-type) inverts the fim switch only from ON to OFF.
   　The results of positive control 1 and negative control 1 confirmed that the endogenous FimB and FimE did not invert our fim switch (wild-type). Also, the result of positive control 2 and negative control 2, indicates that the expression of FimE (wild-type) do not have effects on the GFP expression.

Fig. 3-4-3-1. Histogram of the samples measured by flow cytometer

3.2. FLA analysis

　　　　　　

写真とシークエンスデータ

4. Discussion

When the concentration of FimB (wild-type) increased by increasing the concentration of arabinose, we confirmed that the fluorescence intensity decreased in both ON to OFF process and OFF to ON process.    The result of the reporter cell (2) shows that when the concentration of arabinose is increased to 0〜20 microM, the fluorescence intensity increases. This shows the function of FimB (wild-type) inverting the fim switch (wild-type) from OFF to ON. However, when the arabinose concentration is excess amount, (5mM)、the fluorescence intensity decreases (Fig.3-5-4-1). According to [1], this is caused by the excess increase of the inversion rate of the fim switch. When the inversion rate is too high, there is not enough time for transcription initiation. Consequently, the GFP expression decreases.

Fig.3-4-4-1. Histogram of reporter cell (2)

	Even though there is no fim switch (wild-type) in the plasmid of positive control 2, similar increase of fluorescence intensity dependent on the expression of FimB (wild-type) was found in our positive control 2 (Fig.3-5-4-1) This unpredictable increase of fluorescence intensity is caused by the decrease of dilution rate of proteins inside cells. The FimB (wild-type) expression, depending on the arabinose induction, inhibits cell division that decreases protein concentration inside the individual cells. Therefore, the concentration of GFP in individual cell increases.
Fig.3-4-4-2. The histogram of positive control 2

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