contents

1. Introduction

2. Summary of the Experiment

3. Results

3.1. Arabinose dependent FimE expression

3.2. FLA analysis

4. Materials and Methods

5. Materials and Methods

5.1. Construction

5.2. Assay Protocol

5.2.1. C4HSL-dependent CmR expression assay

5.2.2. C4HSL-dependent CmR expression assay (With an ssrA tag)

5.2.3. Chloramphenicol-dependent Growth Assay with ssrA tag

5.2.4. C4HSL-dependent CmR expression assay ([Cm] = 75 microg/mL)

6. Reference

1. Introduction

　　　　　　

We decided that the fim switch, which the promoter containing repeated DNA sequence can be inverted back and forth at random in the presence of FimB recombinase, is the part we need in order to enable the prisoner coli to make the option between cooperation and defection (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.

　　　　　　

To confirm the function of the newly constructed plasmid, P_BAD/araC_fimB (BBa_K1632012), we also constructed two 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 the fim switch [default ON] or the fim switch [default OFF] is placed upstream of the GFP coding sequence.

　　　　　　

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

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 JM2.300 strain.

-Plasmids

　　　　　　

1. Pcon_rhlR_TT_Plux_CmRssrA(pSB6A1) + Plac_lasI(pSB3K3)

Fig. 3-2-5-1.

　　　　　　

2. Pcon_rhlR_TT_Plux_CmRssrA(pSB6A1) +promoter less_lasI(pSB3K3)

Fig. 3-2-5-2.

　　　　　　

3. Pcon_rhlR_TT_Plux_CmR(pSB6A1) + Plac_lasI(pSB3K3)

Fig. 3-2-5-3.

　　　　　　

4. Pcon_rhlR_TT_Plux_CmR(pSB6A1) +promoter less_lasI(pSB3K3)

Fig. 3-2-5-4.

　　　　　　

5. Negative control1: Pcon_rhlR_TT_promoter less_CmR (pSB6A1) + Plac_lasI (pSB3K3)

Fig. 3-2-5-5.

　　　　　　

6. Neative cotrol2:Pcon_rhlR_TT_promoter less_CmR (pSB6A1) +promoter less_lasI (pSB3K3)

Fig. 3-2-5-6.

5.2. Assay Protocol

5.2.1. C4HSL-dependent CmR expression assay

-samples
Pcon_rhlR_TT_Plux_CmR(pSB6A1)＋Plac_lasI(pSB3K3)#1
Pcon_rhlR_TT_Plux_CmR(pSB6A1)＋Plac_lasI(pSB3K3)#2
Pcon_rhlR_TT_Plux_CmR(pB6A1)＋ promoter lesslasI(pSB3K3)#1
Pcon_rhlR_TT_Plux_CmR (pSB6A1) ＋ promoter less_lasI (pSB3K3)#2
Pcon_rhlR_TT_promoter less_CmR (pSB6A1)＋promoter less_lasI(pSB3K3)#1
Pcon_rhlR_TT_promoter less_CmR (pSB6A1)＋promoter less_lasI(pSB3K3)#2
Pcon_rhlR_TT_promoter less_CmR (pSB6A1)＋promoter less_lasI(pSB3K3)#1
Pcon_rhlR_TT_promoter less_CmR (pSB6A1)＋promoter less_lasI(pSB3K3)#2

-Procedure
1. Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.
3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
4. Suspend the pellet in 1mL of LB containing Amp and Kan.
5. Add 30 microL of suspension in the following medium.
   ①)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microM C4HSL (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)
   ②)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)
6. Grow the samples of cells at 37°C for more than 8 hours.
7. Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/5.)

5.2.2. C4HSL-dependent CmR expression assay (With an ssrA tag)

-samples
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI(pSB3k3)#1
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI(pSB3k3)#2
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI(pSB3k3)#1
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI(pSB3k3)#2
Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋Plac_lasI (pSB3k3)#1
Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋Plac_lasI(pSB3k3)#2
Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋promoter less_lasI(pSB3k3)#1
Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋promoter less_lasI(pSB3k3)#2
Pcon_rhlR_TT_promoter less _CmR(pSB6A1)＋Plac_lasI(pSB3k3)#1
Pcon_rhlR_TT_promoter less _CmR(pSB6A1)＋Plac_lasI(pSB3k3)#2
Pcon_rhlR_TT_promoter less _CmR(pSB6A1)＋promoter less_lasI(pSB3k3)#1
Pcon_rhlR_TT_promoter less _CmR(pSB6A1)＋promoter less_lasI(pSB3k3)#2

-Procedure
1. Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.
3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
4. Suspend the pellet in 1mL of LB containing Amp and Kan.
5. Add 30 microL of suspension in the following medium.
   ①)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microM C4HSL (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)
   ②)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)
6. Grow the samples of cells at 37°C for more than 8 hours.
7. Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/5.)

5.2.3. Chloramphenicol-dependent Growth Assay with ssrA tag

-samples
Pcon_rhlR_TT_Plux_CmRssrA(pSB6A1)＋Plac_lasI(pSB3k3)#1 Pcon_rhlR_TT_Plux_CmRssrA(pSB6A1)＋Plac_lasI(pSB3k3)#2 Pcon_rhlR_TT_Plux_CmRssrA(pSB6A1)＋promoter less_lasI(pSB3k3)#1 Pcon_rhlR_TT_Plux_CmRssrA(pSB6A1)＋promoter less_lasI(pSB3k3)#2 Procedure
1. Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.
3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
4. Suspend the pellet in 1 mL of LB containing Amp and Kan.
5. Add 30 microL of suspension in the following medium.
   ①) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (6 microL of 25 microg/mL) + 99.5% ethanol (6 microL)
   ②) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (9 microL of 25 microg/mL) + 99.5% ethanol (3 microL)
   ③) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (12 microL of 25 microg/mL)
6. Grow the samples of cells at 37°C for more than 8 hours.
7. Measure the optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)

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. Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619