contents

1. Introduction

2. Summary of the Experiment

3. Results

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

3.2. Supplemental experiments

4. Discussion

5. Materials and Methods

5.1. Construction

5.2. Assay Protocol

5.2.1 Arabinose dependent FimB(wile-type) expression

5.2.2. Supplemental experiments

6. Reference

1. Introduction

In order to enable a prisoner coli to randomly select its option between cooperation and defection, we noticed that a fim switch(wild-type), which can invert a promoter sequence bidirectionally in the presence of FimB(wild-type) recombinase, is the part we need (Fig. 3-4-1-1).
	Fig. 3-4-1-1. In the presence of FimB(wild-type) recombinase, the fim switch which is a promoter containing repeated DNA sequence, is inverted at random.

For implementation of Decision making coli, we newly constructed plasmid, P_BAD/araC_fimB(wild-type) (BBa_K1632012) that produces FimB(wild-type). We also prepared two other new plasmids, BBa_K1632007 and BBa_K1632008 (Fig. 3-4-1-2). BBa_K1632012 enables arabinose-inducible expression of FimB(wild-type). In BBa_K1632007 and BBa_K1632008, either [ON] or [OFF] fim switch(wild-type) is placed upstream of GFP coding sequence.

Fig.3-4-1-2. New plasmids we constructed to confirm the function of BBa_K1632012 plasmid for Decision making coli.

2. Summary of the Experiment

Our purpose is to confirm that FimB(wild-type) inverts the fim switch(wild-type) from [ON] state to[OFF] state and from [OFF] state to [ON] state (Fig.3-4-2-1). We prepared six plasmids below. 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] state to [OFF] state and [OFF] state to [ON] state. From the results we also confirmed our fim switch(wild-type) is not inverted by the endogenous FimB and FimE and that FimB(wild-type) expression doesn’t affect the GFP expression. In order to confirm inversion more precisely, we also show the percentage of [ON] state with induction by arabinose and without induction. Also we show inversion in the level of DNA sequencing.

(1) P_BAD/araC_fimB(wild-type) (pSB6A1) + fim switch[default ON](wild-type)_gfp (pSB3K3)
(2) P_BAD/araC_fimB(wild-type) (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_fimB(wild-type) (pSB6A1) + Pcon_gfp (pSB3K3)
(6) Negative control 2 : P_BAD/araC_fimB(wild-type) (pSB6A1) + promoter less_gfp (pSB3K3)

Fig.3-4-2-1. Plasmids for the experiment of FimB dependent fim switch state assay

3. Results

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

We tried to confirm that the fim switch is bidirectically inverted in the presence of FimB(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 0.5 percent glucose in order to repress the leakage in the P_BAD/araC promoter. Fig. 3-4-3-1 shows the histograms of the samples measured by the flow cytometer. In the results of the reporter cell (1), when the induction of FimB(wild-type) expression increases, the fluorescence intensity decreases. From this fact, we confirmed that the fim switch(wild-type) is inverted from [ON] state to [OFF] state by FimB(wild-type). From the result of the reporter cell (2), when the expression amount of FimB(wild-type) increases, the expression amount of GFP in the reporter cell (2) increases. From this fact, we confirmed that the fim switch(wild-type) is inverted from [OFF] state to [ON] state by FimB(wild-type). From the results of the two reporter cells (1) and (2), we successfully confirmed that FimB(wild-type) inverts the fim switch(wild-type) from [ON] state to [OFF] state and from [OFF] state to [ON] state.
   　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 Negative control 2, indicates that the expression of FimB(wild-type) do not affect GFP expression. The reason the fluorescence intensity of the Positive control 2 is increasing in proportion to the arabinose concentration is described in 4. Discussion section.

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

3.2. Supplemental experiments

To confirm our results that our FimB(wild-type) inverted the fim switch(wild-type) further, after scattering the samples on a plate, we counted the number of colonies in which GFP is expressed and the colonies which GFP is not expressed. After measurement of flow cytometer, we minipreped the cells culture of leftover and got plasmid mixture which contain pSB6A1 and pSB3K3 in each samples. For example, in sample (1), P_BAD/araC_fimB(wild-type) (pSB6A1) + fim switch[default ON](wild-type)_gfp (pSB3K3), pSB6A1 represents P_BAD/araC_FimB and pSB3K3 represent fim swtich[default ON](wild-type)_gfp and fim switch[default OFF](wild-type)_gfp. E. coli DH5alpha was transformed with the plasmid mixture and cultured on LB plate containing kanamycin and glucose. As a result, E. coli which at least have pSB3K3 containing fim switch[default ON]_gfp or fim switch[default OFF]_gfp form colonies. Some colonies may have both pSB3K3 and pSB6A1 plasmid. However, the fim switch(wild-type) is not inverted by the leak of recombinase because glucose repress araC that activates expression of recombinase.

Fig. 3-4-3-2.  Determination of percentage of [ON] state and colony formation using plasmid mixture extracted cell expressing FimB.

The state of fim switch either [ON] or [OFF] in colonies is evaluated from fluorescence. Thus, colonies which contain fim switch[default ON] expressed GFP. On the other hand, colonies which contain fim switch[default OFF] do not express GFP. Fluorescence intensity were measured by plate reader. We also counted out the all colonies and those with fluorescence. In the results of the reporter cell (1), when inducing the expression of FimB(wild-type), the percentage of [ON] state decreased. Furthermore, from the results of the reporter cell (2), when inducing the expression of FimB(wild-type), the percentage of [ON] state increased. From the results of the two reporter cells (1) and (2), we successfully confirmed that the fimB protein inverts the fim switch(wild-type) from [ON] state to [OFF] state and from [OFF] state to [ON] state (Fig. 3-4-3-2). This result was consistent with the histograms (Fig. 3-4-3-3).

Fig. 3-4-3-3. DNA sequencing results of fim switch.

Also, we incubated the colonies with fluorescence and those without fluorescence. We minipreped cell culture and asked DNA sequencing of each samples. Sequence complementarity in the specific region of the switch shows intended inversion of the switch from [ON] state to [OFF] state in all samples (Fig. 3-4-3-3).

4. Discussion

When the concentration of FimB(wild-type) increased by increasing 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] state to [ON] state. However, when the arabinose concentration is excess (5mM), the fluorescence intensity decreases (Fig. 3-4-4-1). According to [1], this is caused by the excess increase in the inversion rate of the fim switch(wild-type). When the inversion rate is too high, there is not enough time for transcription initiation. Consequently, GFP expression decreases.

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

	Even though there is no fim switch(wild-type) in the plasmid of Positive control 2, similar increase in fluorescence intensity dependent on the expression of FimB(wild-type) was found in our Positive control 2 (Fig. 3-4-4-2) This unpredictable increase in fluorescence intensity is caused by the decrease of dilution rate of proteins inside cells. The FimB(wild-type) expression, dependent 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

5. Materials and Methods

5.1. Construction

-Strain

All the samples were DH5alpha strain.

-Plasmids

(1) P_BAD/araC_fimB(wild-type) (pSB6A1) + fim switch[default ON](wild-type)_gfp (pSB3K3)

Fig. 3-4-5-1.

(2) P_BAD/araC_fimB(wild-type) (pSB6A1) + fim switch[default OFF](wild-type)_gfp (pSB3K3)

Fig. 3-4-5-2.

(3) Posigive control 1 : (pSB6A1) + fim switch[default ON](wild-type)_gfp(pSB3K3)

Fig. 3-4-5-3.

(4) Negative control 1 : (pSB6A1) + fim switch[default OFF](wild-type)_gfp(pSB3K3)

Fig. 3-4-5-4.

(5) Positive control 2 : P_BAD/araC_fimB(wild-type) (pSB6A1) + Pcon_gfp (pSB3K3)

Fig. 3-4-5-5.

(6) Negative control 2 : P_BAD/araC_fimB(wild-type) (pSB6A1) + promoter less_gfp (pSB3K3)

Fig. 3-4-5-6.

5.2. Assay Protocol

5.2.1. Arabinose dependent FimB(wile-type) expression

1. Prepare overnight cultures for each sample in 3 mL of LB medium containing ampicillin (50 microg / mL), kanamycin (30 microg / mL) and glucose (final concentration is 0.5 percent) at 37ºC, shaking at 180 rpm for 12h.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration is 0.5 percent).
3. Incubate the cells at 37ºC, shaking at 180 rpm until the observed OD590 reaches 0.4 (Fresh Culture).
4. After the incubation, take 1 mL of the samples, and centrifuge at 5000x g, 1 min, 25ºC.
5. Remove the supernatant.
6. Suspend 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25ºC.
7. Remove the supernatant.
8. Suspend the pellet in 1 mL of LB containing Amp and Kan, and centrifuge at 5000x g, 1 min, 25ºC.
9. Remove the supernatant.
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, glucose (final concentration is 0.5 percent) and 30 microL of sterile water.
   ②) 3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)
   ③) 3 mL of LB containing Amp, Kan and 30 microL of 20 mM arabinose (final concentration of arabinose is 200 microM)
   ④) 3 mL of LB containing Amp, Kan and 30 microL of 500 mM arabinose (final concentration of arabinose is 5 mM)
   ※ As for (3) and (4), the suspension were added only in medium ① and ④.
12. Incubate the samples at 37ºC, shaking at 180 rpm for 6.5 hours. (Measure OD590 of all the samples every hour.)
13. After the incubation, take the samples, and centrifuge at 9000x g, 1min, 4ºC.
14. Remove the supernatant.
15. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3)
16. Dispense all of each suspension into a disposable tube through a cell strainer.
17. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)

5.2.2. Supplemental experiments

1. After the assay of “Arabinose dependent FimE expression”, miniprep cell culture ((1)-①, (1)-③, (2)-①　and　(2)-③) of leftover as here.
2. Turn on water bath to 42ºC.
3. Take competent DH5alpha strain from -80ºC freezer and leave at rest on ice.
4. Add 3 microL of each plasmids in a 1.5 mL tube.
5. Put 25 microL 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 42ºC for 30 seconds.
8. Put tubes back on ice for 2 minutes.
9. Add 125 microL of SOC medium. Incubate tubes for 30 minutes at 37ºC.
10. Make a 1:5 dilution in 150microL of fresh SOC medium.
11. Spread about 100 microL of the resulting culture of LB plate containing kanamycin.
12. Incubate LB plate for 14-15 hours at 37ºC.
13. Set the plate reader to measure GFP.
14. Scan the each plates with the plate reader. (We used FujiFilm FLA-5100 Fluorescent Image Analyzer from FUJIFilm Life Science.)
15. Analyze the scanning data by changing the scale type (Bezier) and adjusting the range. (We analyzed by using the software, Multi Gauge ver. 2.0 from FUJIFilm Life Science.)
16. Counted out the all colonies and those with fluorescence.
17. Prepare three overnight cultures for each sample in 3 mL of LB medium containing kanamycin (30 microg / mL) shaking at 180 rpm for 12h.
18. Miniprep each samples and ask DNA sequencing of each samples for Biomaterial Analysis Center, Technical Department.

6. Reference

1. Hung M. et al. (2014) Modulating the frequency and bias of stochastic switching to control phenotypic variation. Nat Commun 5:4574. doi:10.1038/ncomms5574

2. Timothy S. Ham et al. (2006) A Tightly Regulated Inducible Expression System Utilizing the fim Inversion Recombination Switch. Biotechnol Bioeng 94(1):1-4