Team:Tokyo Tech/Experiment/C4HSL-dependent growth assay

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C4HSL-dependent CmR expression

　　

contents

1. Introduction

2. Summary of the Experiment

2.1. C4HSL-dependent CmR expression

2.2. Insertion of an ssrA degradation tag to CmR

2.3. Realizing the payoff matrix

2.3.1. Determining the ideal Cm Concentration

2.3.2. Payoff matrix with the lower Cm Concentration

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

　　　　　　

　　　

2. Summary of the Experiment

2.1. C4HSL-dependent CmR expression

　　　　　　

Fig. 3-1-2-1. C4HSL-dependent CmR expression

We confirmed the function of C4HSL-dependent CmR expression by measuring the optical density of the cultures containing chloramphenicol (Cm) (Fig. 3-1-2-1.). In this experiment we prepared four cells which contain different sets of plasmids, (1), (2), (3), and (4) (Fig. 3-1-2-2.). C4HSL and chloramphenicol was added into the medium containing the cells. The optical density was measured every hour for eight hours to estimate the concentration of the cell. (1), and (2) are the cooperating and defecting prisoner coli A, respectively. (3), and (4) are the negative control for (1), and (2), respectively.

　　　　　　

Fig. 3-1-2-2. Plasmids for the experiment of C4HSL-dependent CmR expression

2.2. Insertion of an ssrA degradation tag to CmR

At the first stage of wet experiment, Prisoner cell A and B, which are the initially designed circuits showed leaky expression of CmR. Cells showed active growth even in the absence of AHL when the cell harboring the pairs of plasmids (1) and (2) in Prisoner coli A (Fig. 3-1-2-2.). As a result of our modeling, the influence of the leakage was not reduced by increasing the Cm concentration, which was one of our solutions. (link to modelingリンクさせる！！)
   For precise implementation of our payoff matrix, suggestions from modeling (link to modelingリンクさせる!!) allowed us to successfully solve the influence of the leakage by adding an ssrA tag right after the CmR gene (Pcon_rhlR_TT_Plux_CmRssrA, BBa_K1632023) (Fig. 3-1-2-3.). Protein with an ssrA tag is easily degraded by ClpXP and ClpAP that E.coli originally have. The optical density was measured every hour for eight hours to estimate the growth of the Prisoner coli with the improved parts (Pcon_rhlR_TT_Plux_CmRssrA).

　　　　　　

Fig. 3-1-2-3.  The improved parts, BBa_K1632023, we constructed

　　　　　　

Fig. 3-1-2-4. Cells for the experiment to measure C4HSL-dependent CmR expression

2.3. Realizing the payoff matrix

2.3.1. Determining the ideal Cm Concentration

Using the improved plasmid we constructed, our E.coli version payoff matrix was replicated through wet experiments. The order of the ODs were as expected. (もしくは We tried to realize the payoff matrix.) However, from the results, the difference between “middle” and “high” growth inhibition was hardly observable.
    The growth rate of the Prisoner cells (5) and (6), grown in different Cm concentration (50, 75, 100microg/mL) without C4HSL, were observed. (refer protocol 4-2-3)

2.3.2. Payoff matrix with the lower Cm Concentration

Using the lower Cm concentration (75microg/mL), the growth of the Prisoner cells (5) and (6) were measured to realize the payoff matrix.

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. 3-1-4-1.

　　　　　　

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

Fig. 3-1-4-2.

　　　　　　

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

Fig. 3-1-4-3.

　　　　　　

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

Fig. 3-1-4-4.

　　　　　　

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

Fig. 3-1-4-5.

　　　　　　

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

Fig. 3-1-4-6.

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 (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.)

4.2.2. 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