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. C4HSL-dependent CmR expression assay

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

4.2.3. Chloramphenicol-dependent Growth Assay with ssrA tag

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

5. Reference

1. Introduction

　　　　　　

We designed a signal-dependent growth system by using signaling molecules and antibiotic resistance gene. In our prisoner’s dilemma game, our prisoner coli A needs 3OC12HSL to acquire chloramphenicol resistance (CmR). Pcon_rhlR_TT_Plux_CmR (6A1) cell is an engineered E. coli that contains C4HSL-dependent chloramphenicol resistance gene product (CmR) generator and a constitutive RhlR generator. As a constitutive 3OC12HSL production module, we used Plac_lasI (pSB3k3). For construction of the C4HSL-dependent chloramphenicol resistance gene product (CmR) and 3OC12HSL production module (Plac_lasI), we constructed an improved parts Pcon_rhlR_TT_Plux_CmRssrA ,BBa_K1632023. The C4HSL-dependent growth was confirmed by measuring the optical density.

　　　　　　

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. Materials and Methods

4.1. Construction

-Strain

　　　　　　

All the samples were JM2.300 strain.

-Plasmids

　　　　　　

(1) Pcon_rhlR_TT_Plux_CmRssrA(pSB6A1) + Plac_lasI(pSB3K3)

Fig. 3-2-4-1.

　　　　　　

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

Fig. 3-2-4-2.

　　　　　　

(3) Pcon_rhlR_TT_Plux_CmR(pSB6A1) + Plac_lasI(pSB3K3)

Fig. 3-2-4-3.

　　　　　　

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

Fig. 3-2-4-4.

　　　　　　

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

Fig. 3-2-4-5.

　　　　　　

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

Fig. 3-2-4-6.

4.2. Assay Protocol

4.2.1. 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.)

4.2.2. 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.)

4.2.3. 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.)

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

-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 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 (75 microg/mL)
   ②) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (75 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 0.9, dilute the cell medium to 1/5.)

6. Reference

　　　　　　

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