3OC12HSL-dependent_growth_assay

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 B needs C4HSL to acquire chloramphenicol resistance (CmR).

For construction of the 3OC12HSL-dependent chloramphenicol resistance gene product (CmR) and C4HSL production module, we constructed an improved parts Pcon_lasR_TT_Plux_CmRssrA (BBa_K1632022). In our story, we confirmed the 3OC12HSL-dependent growth by measuring the optical density.

Fig.3-2-1-1. Payoff matrix of Prisoner coli B

2. Summary of the Experiment

2.1 3OC12HSL-dependent CmR expression

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Fig.3-2-2-1. 3OC12HSL-dependent CmR expression

We confirmed the function of 3OC12HSL-dependent CmR expression by measuring optical density of the cultures containing chloramphenicol (Fig. 3-2-2-1).In this experiment we prepared four plasmids, A, B, C, and D (Fig. 3-2-2-2). Right after the 3OC12HSL induction, we added chloramphenicol into the medium containing Prisoner cell. We measured the optical density for about eight hours to estimate the concentration of the cell.

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Fig.3-2-2-2. Plasmids for the experiment of 3OC12HSL-dependent CmR expression

2.3 Adding an ssrA degradation tag

At the first stage of wet experiment, initially designed circuits showed leaky expression of CmR. Although “middle” and “high” growth inhibition is required for implementation of our payoff matrix (Fig. X), cells showed active growth even in the absence of AHL when the cell harboring our initially designed genetic circuit Pcon_lasR_TT_Plux_CmR in Prisoner coli B (Fig. X). We could not obtain positive results in our modeling by increasing the concentration of Cm, which was one of our solutions. For precise implementation of our payoff matrix, suggestions from modeling allow us successfully improving the former plasmid by adding an ssrA tag right after the CmR gene (Pcon_lasR_TT_Plux_CmRssrA (BBa_K1632022)) (Fig.3-2-2-3). The ssrA tag helps to degrade the leaked CmR protein. The improved parts were used for construction of BBa_K1632022 circuit for C4HSL inducible expression of CmR. Compared with circuits without ssrA tag BBa_K395160, our improved BBa_K1632022 indeed showed much slower growth which corresponds to “middle” growth inhibition (Fig.3-2-2-1). Furthermore, addition of C4HSL recovers active cell growth which corresponds to “none” growth inhibition (Fig. 2-1). These results show the improved function of AHL-dependent CmR expression by measuring the optical density. (Projectより引用)

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Fig.3-2-2-3. The improved plasmid (BBa_K1632022) we constructed

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Fig.3-2-2-4. Plasmids for the experiment of 3OC12HSL-dependent CmR expression

2.3 Realizing the payoff matrix

2.3.1 Seeking the ideal Cm Concentration

Using the improved plasmids we constructed, our E.coli version payoff matrix is replicated through wet lab experiments. However, from the results shown in (Fig. X), the difference between “middle” growth inhibition and “high” growth inhibition was hardly observable.

The experiment was conduvted with different Chloramphenicol concentration (50, 75, 100microg/mL). Incubated in a culture medium without AHL, the difference in the growth rate was observed between the one producing AHL and the one not.

2.3.2 Payoff matrix with the new Cm concentraion

We found out that 75microg/mL is a Cm concentration good enough to realize the payoff matrix precisely. Using the new Cm concentration (75microg/mL), the “C12HSL-dependent CmR expression assay” (refer to 5-2-4) was run again to replicate a precise payoff matrix.

2.4 Decreasing the AHL(3OC12HSL) Concentration

3. Results

3.1 3OC12HSL-dependent CmT expression

We tested four types of culture condition which contains different Cm concentration (0 and 100 microg/mL) and different AHL concentration (0 and 5 nM). Fig. 3-2-3-1, Fig. 3-2-3-2, Fig. 3-2-3-3, Fig. 3-2-3-4 show the condition in the absence and presence of Cm, respectively. Regardless of the presence of Cm, every cell grew in the culture medium even without 3OC12HSL.

Cm (+)…Pcon_lasR_TT_Plux_CmR (6A1) + Plac_rhlI (3K3) (8/27, 28)

Fig.3-2-3-1. Cooperating Prisoner coli B’s growth with Cm

Cm (+)…Pcon_lasR_TT_Plux_CmR (6A1) + ⊿P_rhlI (3K3) (8/27,28)

Fig.3-2-3-2. Defecting Prisoner coli B’s growth with Cm

The expression and the function of CmR was confirmed from (Fig3-2-3-1.) and (Fig. 3-2-3-2), since the Prisoner coli have grown (refer the solid magenta line of (Fig3-2-3-1.) and (Fig. 3-2-3-2)) despite the presence of chloramphenicol in the culture. However, the Prisoner coli have also grown in the culture with chloramphenicol without AHL (dotted magenta line), in other words, the prisoner coli have acquired Cm resistance regardless of the presence of 3OC12HSL. From this fact, leakage in the promoter was suspected (assumed) ((Fig3-2-3-1.) & (Fig. 3-2-3-2)).

3.2 Plasmids with an ssrA degradation tag

We repeated the experiment (refer to 3.1) using the new plasmid we constructed. From the results of our experiment, we confirmed that the new prisoner coli B (Pcon_lasR_TT_Plux_CmRssrA) had expressed CmR when induced by 3OC12HSL, as expected (Fig. 3.2.1 and Fig. 3.2.2).

Pcon_lasR_TT_Plux_CmR (6A1) + Plac_rhlI (3K3) (8/27, 28)

Fig. 3-2-3-3. Cooperating Prisoner coli B’s growth with an ssrA tag

Pcon_lasR_TT_Plux_CmR (6A1) + ⊿P_rhlI (3K3) (8/27, 28)

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Fig.3-2-3-4. Defecting Prisoner coli B's growth with an ssrA tag

Protein with an ssrA tag is said to be easy to be dissolved by ClpXP and ClpAP that E.coli originally have. From (Fig.3-2-3-3) and (Fig.3-2-3-4), Prisoner coli with an ssrA tag were not able to grow without 3OC12HSL. Therefore, we can say that CmR produced by the leak of the Plux promoter was dissolved immediately while an ssrA tag was added to CmR. Adding an ssrA tag can be said a sufficient method to reduce the influence of the leak of Plux promoter.

Comparing the growth in the 3CO12HSL lacking culture medium of the initial prisoner coli and the new prisoner coli with the ssrA tag, in other words the magenta dotted line and the green dotted line of each (Fig. 3-2-1) and (Fig. 3-2-2), showed that the leaky CmR was reduced by adding an ssrA tag.

3.3 Realizing the payoff matrix

3.3.1 Seeking the ideal Cm concentration

We ran the experiment with different Chloramphenicol concentration (50, 75, 100 microg/mL).The following three results are the OD of cooperating Prisoner B (Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) + Plac_rhlI (pSB3K3)) and defecting Prisoner B (Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) +⊿P_rhlI (pSB3K3)) grown in the culture medium without 3OC12HSL. The growth inhibition degree of each stand for “high” and “middle” growth inhibition.

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Fig.3-2-3-5

We tried to make the growth inhibition rate of chloramphenicol larger than the metabolic burden of producing C4HSL. But at the same time, making the difference between “middle” and “high” growth inhibition, in other words, replicating a precise pay off matrix, is also our goal. From the experimental results, 75 microg/mL was determined to be a Cm concentration good enough to realize the precise payoff matrix, while the green lines and the orange lines in (Fig. 3-2-3-5) were able to distinguish.

3.3.2 Payoff matrix with the new Cm Concentration

We ran the “3OC12HSL-dependent CmR expression assay” with the new Chloramphenicol concentration (75 microg/mL). The results are the following.

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Fig.3-2-3-6

With the new Chloramphenicol Concentration, the payoff matrix was replicated precisely. The orange line is when you cooperate, and the green is when you defect. The solid line is when your opponent cooperates, and the dotted is when your opponent defects.

3.4 Decreasing the AHL(3OC12HSL) Concentration

4. Materials and Methods

4.1. Construction

-Strain

All the samples were JM2.300 strain.

-Plasmids

(1) Pcon_lasR_TT_Plux_CmR (pSB6A1) + Plac_rhlI (pSB3K3)


Fig. 3-2-4-1.

(2) Pcon_lasR_TT_Plux_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)


Fig. 3-2-4-2.

(3) Pcon_lasR_TT_Plux_CmR (pSB6A1) + Plac_rhlI (pSB3K3)


Fig. 3-2-4-3.

(4) Pcon_lasR_TT_Plux_CmR (pSB6A1) +promoter less_rhlI (pSB3K3)


Fig. 3-2-4-4.

(5) Negative control1: Pcon_lasR_TT_promoter less_CmR (pSB6A1) + Plac_rhlI (pSB3K3)


Fig. 3-2-4-5.

(6) Negative cotrol2:Pcon_lasR_TT_promoter less_CmR (pSB6A1) +promoter less_rhlI (pSB3K3)


Fig. 3-2-4-6.

4.2. Assay Protocol

4.2.1. 3OC12HSL-dependent CmR expression assay

-samples
Pcon_lasR_TT_Plux_CmR (pSB6A1) + Plac_rhlI (pSB3K3)　#1
Pcon_lasR_TT_Plux_CmR (pSB6A1) + Plac_rhlI (pSB3K3)　#2
Pcon_lasR_TT_Plux_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)　#1
Pcon_lasR_TT_Plux_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)　#2
Pcon_lasR_TT_promoter less _CmR (pSB6A1) + Plac_rhlI (pSB3K3)　#1
Pcon_lasR_TT_promoter less _CmR (pSB6A1) + Plac_rhlI (pSB3K3)　#2
Pcon_lasR_TT_promoter less _CmR (pSB6A1) + promoter less_rhlI (pSB3K3)　#1
Pcon_lasR_TT_promoter less _CmR (pSB6A1) + promoter less_rhlI (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) + 5 microM 3OC12HSL (3 microL) + Chloramphenicol (3 microL of 100 microg/mL)
②)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 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 0.9, dilute the cell medium to 1/5.)

4.2.2. 3OC12HSL-dependent CmR expression assay with an ssrA tag

-samples
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1)＋Plac_rhlI (pSB3K3)#1
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1)＋Plac_rhlI (pSB3K3)#2
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_rhlI (pSB3K3)#1
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_rhlI (pSB3K3)#2
Pcon_lasR_TT_Plux_CmR (pSB6A1)＋Plac_rhlI (pSB3K3)#1
Pcon_lasR_TT_Plux_CmR (pSB6A1)＋Plac_rhlI (pSB3K3)#2
Pcon_lasR_TT_Plux_CmR (pSB6A1)＋promoter less_rhlI (pSB3K3)#1
Pcon_lasR_TT_Plux_CmR (pSB6A1)＋promoter less_rhlI (pSB3K3)#2
Pcon_lasR_TT_promoter less _CmR (pSB6A1)＋Plac_rhlI (pSB3K3)#1
Pcon_lasR_TT_promoter less _CmR (pSB6A1)＋Plac_rhlI (pSB3K3)#2
Pcon_lasR_TT_promoter less _CmR (pSB6A1)＋promoter less_rhlI (pSB3K3)#1
Pcon_lasR_TT_promoter less _CmR (pSB6A1)＋promoter less_rhlI (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) + 5 microM 3OC12HSL (3 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 an ssrA tag

-samples
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1)＋Plac_rhlI (pSB3K3)#1
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1)＋Plac_rhlI (pSB3K3)#2
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_rhlI (pSB3K3)#1
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_rhlI (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. 3OC12HSL-dependent CmR expression assay ([Cm] = 75 microg/mL)

-Samples
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1)＋Plac_rhlI (pSB3K3)#1
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1)＋Plac_rhlI (pSB3K3)#2
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_rhlI (pSB3K3)#1
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_rhlI (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) + 5 microM 3OC12HSL (3 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

Team:Tokyo Tech/Experiment/3OC12HSL-dependent growth assay