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

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FimB dependent fim switch state_assay

We have characterized previous parts.

　　

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

　　　　　　

We decided that fim switch, which can invert promoter back and forth at random in the presence of FimB recombinase, is the part we need in order to enable prisoner coli to make option between cooperation and defection (Fig. 3-4-1-1).

　　　　　　

Fig.3-4-1-1. In the presence of FimB recombinase, promoter fim switch can invert at random.

　　　　　　

For the confirmation of the function of newly constructed plasmid, PBAD/araC_rbs_fimB (BBa_K1632012), we also prepared two new plasmids, BBa_K1632007 and BBa_K1632008 (Fig. 2.9). BBa_K1632012 enables arabinose-inducible expression of wild type FimB. In BBa_K1632007 and BBa_K1632008, either [ON] or [OFF] fim switch is placed upstream of GFP coding sequence.

　　　　　　

Fig.3-4-1-2. New plasmids we constructed to confirm the function of fim switch in the FimB presence

2. Summary of the Experiment

　　　　　　

Our purpose is to confirm that FimB inverts fimswitch from ON to OFF and OFF to ON (図の番号). Taking endogenous FimB and FimE into account, we prepared six plasmids sets shown in below(図の番号). 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った。

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

3. Results

3.1. Arabinose dependent FimE expression

　　　　　　

私たちは、4種類のarabinose濃度でFimBが働くかどうかを、GFPを用いたレポーターアッセイによって確かめた。 　Figure(図番号) は、default ONのサンプルが、arabinose誘導によって、OFF状態に切り替わった結果を示している。 またFigure(図番号)は、default OFFのサンプルが、arabinose誘導によって、ON状態に切り替わった結果を示している。 Figure(図番号) shows our experimental results of FimB and Fimswitch. From the results of the reporter cell C and D, inversion from ON to OFF and OFF to ON by endogenous proteins are negligible. 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。 以上の2つの結果から、FimBが理想的に両反転を起こしていることがわかる。

Fig. 3-4-3-1.

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

Fig. 3-4-5-1.

　　　　　　

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

Fig. 3-4-5-2.

　　　　　　

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

Fig. 3-4-5-3.

　　　　　　

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

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 FimE 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