FimE dependent fim switch state assay

1. Introduction

To confirm the function of the fim switch in the presence of FimE(wild-type), we constructed two Biobrick parts, BBa_K1632013　(Fig. 3-5-1-1). BBa_K1632013 enables arabinose-inducible expression of the FimE(wild-type). In BBa_K1632007 and BBa_K1632008, 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 BBa_K1632013 plasmid for Decision making coli

2. Summary of the Experiment

Our purpose is to confirm that FimE(wild-type) inverts the fim switch(wild-type) from [ON] state to [OFF] state and from [OFF] state to [ON] state. We prepared six plasmids below (Fig.3-5-2-1). 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 FimE(wild-type) expression doesn’t affect 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_fimE(wild-type) (pSB6A1) + fim switch[default ON](wild-type)_gfp (pSB3K3)
(2) P_BAD/araC_fimE(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_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(wild-type) is predominantly 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 percent glucose in order to repress the leakage in the P_BAD/araC promoter. Fig. 3-5-3-1 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] state to [OFF] state 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] state to [OFF] state 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(wild-type) only from [ON] state to [OFF] 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 Positive control 2 and Negative control 2, indicates that the expression of FimE(wild-type) do not affect GFP expression.


Fig. 3-5-3-1. The histograms of the samples measured by flow cytometer

3.2. Supplemental experiments

To confirm our results that our FimE(wild-type) inverted the fim switch(wild-type) further, after scattering the samples on a plate, we also counted out the all colonies and those with fluorescence. Also we show inversion in the level of DNA sequencing.
After measurement of flow cytometer, we minipreped the cell culture of leftover and got plasmid mixture which contain pSB6A1 and pSB3K3 in each samples. For example, in sample (1), P_BAD/araC_fimE(wild-type) (pSB6A1) + fim switch[default ON](wild-type)_gfp (pSB3K3), pSB6A1 represents P_BAD/araC_fimE 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](wild-type)_gfp or fim switch[default OFF](wild-type)_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. 3-5-3-2. Determination of percentage of [ON] state and colony formation using plasmid mixture extracted cell expressing FimE(wild-type).

The state of fim switch(wild-type) either [ON] state or [OFF] state in colonies is evaluated from fluorescence. Thus, colonies which contain fim switch[default ON](wild-type) expressed GFP. On the other hand, colonies which contain fim switch[default OFF](wild-type) 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 FimE(wild-type), the percentage of [ON] state decreased dramatically. On the other hand, from the results of the reporter cell (2), when inducing the expression of FimE(wild-type), the percentage of [ON] state remained being small. From the results of the two reporter cells (1) and (2), we successfully confirmed that FimE(wild-type) inverts the fim switch(wild-type) predominantly from [ON] state to [OFF] state (Fig. 3-5-3-2). This result was consistent with the histograms (Fig. 3-5-3-1).

Fig. 3-5-3-3. DNA sequencing results of fim switch(wild-type).

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. 3-5-3-3).

4. Materials and Methods

4.1. Construction

-Strain

All the samples were DH5alpha strain.

-Plasmids

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


Fig. 3-5-4-1.

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


Fig. 3-5-4-2.

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


Fig. 3-5-4-3.

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


Fig. 3-5-4-4.

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


Fig. 3-5-4-5.

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


Fig. 3-5-4-6.

4.2. Assay Protocol

4.2.1. Arabinose dependent FimE(wild-type) 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 is 1.0 percent) at 37ºC for 12 h.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp, Kan and glucose (final concentration is 1.0 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 the pellet in 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 1.0 percent) and 30 microL sterile water
   ② 3 mL of LB containing Amp, Kan and 30 microL of 500 microM arabinose (final concentration of arabinose is 5 microM)
   ③ 3 mL of LB containing Amp, Kan and 30 microL of 1 mM arabinose (final concentration of arabinose is 10 microM)
   ④ 3 mL of LB containing Amp, Kan and 30 microL of 2 mM arabinose (final concentration of arabinose is 20 microM)
   ※ As for (3) and (4), the suspension were added only in medium ① and ④.
12. Incubate the samples at 37ºC for 6 hours, shaking at 180 rpm.(Measure the 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.)

4.2.2. 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.(http://parts.igem.org/Help:Protocols/Miniprep)
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 150 microL 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 12 h.
18. Miniprep each samples and ask DNA sequencing of each samples for Biomaterial Analysis Center, Technical Department.

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

Team:Tokyo Tech/Experiment/FimE dependent fim switch state assay

FimE dependent fim switch state assay

contents

1. Introduction

2. Summary of the Experiment

3. Results