FimB dependent fim switch state assay

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 recombinase, the fim switch which is a promoter containing repeated DNA sequence, is invert 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 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-tyoe) 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 FImB expression doesn’t affect 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_fimB (pSB6A1)+ fim switch[default ON](wild-type)_GFP (pSB3K3)
(2) P_BAD/araC_fimB (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 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 % 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 FimB(wild-type) expression increases, the fluorescence intensity decreases. From this fact, we confirmed that the fim switch is inverted from ON to OFF 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 is inverted from OFF to ON 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 from ON to OFF and from OFF to ON. 　The results of positive control 1 and negative control 1 confirmed that the endogenous FimB and FimE did not invert our fim switch (wild-tyoe). Also, the result of negative control 2, indicates that the expression of FimB (wild-type) do not have effects on the 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. 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 FimB concentration increased by increasing arabinose concentration, we confirmed that Fluorescence intensity was decreased in both of ON to OFF and OFF to ON.
According to [1], increasing switching frequency by increasing FimB expression decrease mean expression because it is enough time for FimB to bind to the inversion sequences and disrupt transcription initiation or elongation.
Similar increase dependent on FimB expression was found in control samples(図). Because FimE expression decreases cell growth rate, decreased dilution rate of proteins including GFP from leaky expression in the cells could slightly increase of fluorescence in a cell.

5. Materials and Methods

5.1. Construction

-Strain

All the samples were DH5alpha strain.

-Plasmids

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


Fig. 3-4-5-1.

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


Fig. 3-4-5-2.

C. Posigive control1:(pSB6A1)+ fim switch[default ON](wild-type)_gfp(pSB3K3)


Fig. 3-4-5-3.

D. Negative control2: (pSB6A1)+ fim switch[default OFF](wild-type)_gfp(pSB3K3)


Fig. 3-4-5-4.

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


Fig. 3-4-5-5.

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


Fig. 3-4-5-6.

5.2. Assay Protocol

5.2.1. Arabinose dependent FimB expression

1. Prepare overnight cultures for the each sample in 3 mL of LB medium containing ampicillin (50 microg / mL), kanamycin (30 microg / mL)　and glucose (final concentration of mass of glucose is 0.5 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 0.5 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, glucose (final concentration of mass of glucose 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 C and D, the suspension were added only in medium ① and ④.
12. Grow the samples at 37 ℃ for 6.5 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.)

5.2.2. FLA analysis

1. After the assay of “Arabinose dependent FimE 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℃.

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

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

FimB dependent fim switch state assay

contents

1. Introduction

2. Summary of the Experiment

3. Results