ssrA tag degradation assay

1. Introduction

In order to enable prisoner colis acquire chloramphenicol resistance by receiving the corresponding AHL, we designed a signal-dependent growth system. In our Prisoner’s Dilemma game, Prisoner A coli needs C4HSL and Prisoner B coli needs 3OC12HSL to acquire chloramphenicol resistance. There will be difference in the growth depending on the presence of AHL.
For precise implementation of our payoff matrix, we constructed improved parts Pcon_lasR_TT_Plux_CmRssrA, and Pcon_rhlR_TT_Plux_CmRssrA. The growth of the prisoner colis were confirmed by measuring the optical density.


Fig. 3-1-1-1. The genetic circuits of prisoner colis with the options of cooperation or defection

2. Summary of the Experiment

2.1. AHL-dependent CmR expression

2.1.1. C4HSL-dependent CmR expression (Prisoner A)


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

We confirmed the function of C4HSL-dependent CmR expression parts 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 pairs of plasmids, (1), (2), (3), and (4) (Fig. 3-1-2-2.). The four cells were cultured with or without C4HSL induction. The optical density was measured to estimate the concentration of the cell. Cells containing (1), and (2) are the cooperating and defecting Prisoner A coli, respectively. (3), and (4) are the pairs of plasmids of the negative control for (1), and (2), respectively.


Fig. 3-1-2-2. Pairs of plasmids for the C4HSL-dependent CmR expression assay

2.1.2. 3OC12HSL-dependent CmR expression (Prisoner B)


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

We confirmed the function of 3OC12HSL-dependent CmR expression by measuring the optical density of the cultures containing chloramphenicol (Cm) (Fig. 3-1-2-3.). In this experiment we prepared four cells which contain different pairs of plasmids, (5), (6), (7), and (8) (Fig. 3-1-2-4.). The four cells were cultured with or without 3OC12HSL induction. The optical density was measured to estimate the concentration of the cell. Cells containing (5), and (6) are the cooperating and defecting Prisoner B coli, respectively. (7), and (8) are the pairs of plasmids of the negative control for (5), and (6), respectively.


Fig. 3-1-2-4. Pairs of plasmids for the experiment of 3OC12HSL-dependent CmR expression

2.2. Insertion of an ssrA degradation tag to CmR

At the wet experiment of 2-1., Prisoner colis containing the pairs of plasmids (1) and (2), showed leaky expression of CmR. Cells grew actively even in the absence of AHL when the cell harboring the pairs of plasmids (1) and (2). We came up with two solutions, either increasing the chloramphenicol (Cm) concentration or inserting an ssrA tag to the CmR gene, to this problem. Our modeling result shows that the influence of the leakage was not reduced by increasing the Cm concentration. Please refer to here.

For precise implementation of our payoff matrix, suggestions from modeling allowed us to successfully solve the influence of the leakage by adding an ssrA degradation tag added to the C-terminal of the CmR protein. ((Fig. 3-1-2-5.) and (Fig.3-1-2-6.)) New pairs of plasmids (9), (10), (11) and (12) were prepared as in (Fig. 3-1-2-7.) and (Fig. 3-1-2-8.). The growth of the Prisoner A colis and Prisoner B colis, containing the improved parts, Pcon_rhlR_TT_Plux_CmRssrA (BBa_K1632023), and Pcon_lasR_TT_Plux_CmRssrA (BBa_K1632022), respectively, were measured.


Fig. 3-1-2-5. The improved parts (BBa_K1632023) we constructed

Fig. 3-1-2-6. The improved parts (BBa_K1632022) we constructed


Fig. 3-1-2-7. Pairs of plasmids for the ssrA degradation tag assay of Prisoner A

Fig. 3-1-2-8. Pairs of plasmids for the ssrA degradation tag assay of Prisoner B

3. Results

3.1. AHL-dependent CmR expression

3.1.1. C4HSL-dependent CmR expression (Prisoner A)

The cell growth with and without C4HSL was measured every hour for eight hours. (Fig. 3-1-3-1.) shows the growth of prisoner colis containing the pairs of plasmids (1) and (3). (Fig. 3-1-3-2.) shows the growth of prisoner colis containing the pairs of plasmids (2) and (4). Each Prisoner coli grew in the culture medium even without C4HSL.

Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)


Fig. 3-1-3-1. Cooperating Prisoner A coli’s growth with chloramphenicol

Pcon_rhlR_TT_Plux_CmR (pSB6A1) + promoter less_lasI (pSB3K3)


Fig. 3-1-3-2. Defecting Prisoner A coli’s growth with chloramphenicol

The expression of CmR was confirmed from (Fig. 3-1-3-1.) and (Fig. 3-1-3-2.), since the prisoner coli grew in the presence of chloramphenicol (solid green line of (Fig. 3-1-3-1.) and (Fig. 3-1-3-2.)). However, the Prisoner coli also showed active growth even in the absence of C4HSL (dotted green line), in other words, the prisoner coli acquired Cm resistance independent on C4HSL induction. From this result, leakage in the promoter was expected

3.1.2. 3OC12HSL-dependent CmR expression (Prisoner B)

The cell growth with and without 3OC12HSL was measured every hour for eight hours. (Fig. 3-1-3-3.) shows the growth of prisoner colis containing the pairs of plasmids (5) and (7). (Fig. 3-1-3-4.) shows the growth of prisoner colis containing the pairs of plasmids (6) and (8). Each Prisoner coli grew in the culture medium even without 3OC12HSL.

Pcon_lasR_TT_Plux_CmR (pSB6A1) + Plac_rhlI (pSB3K3)

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

Pcon_lasR_TT_Plux_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)

Fig. 3-1-3-4. Defecting Prisoner B coli’s growth with Cm

The expression of CmR was confirmed from (Fig. 3-1-3-3.) and (Fig. 3-1-3-4.), since the prisoner coli grew in the presence of chloramphenicol (solid green line of (Fig. 3-1-3-3.) and (Fig. 3-1-3-4.)). However, the Prisoner coli also showed active growth even in the absence of 3OC12HSL (dotted green line), in other words, the prisoner coli acquired Cm resistance independent on C4HSL induction. From this result, leakage in the promoter was expected.

3.2. AHL-dependent CmRssrA expression

3.2.1. C4HSL-dependent CmRssrA expression (Prisoner A)

The cell growth of the prisoner colis which contain the pairs of plasmids (1) – (4), (9), and (10) were measured every hour for eight hours. (Fig. 3-1-3-5.) shows the growth of prisoner colis containing the pairs of plasmids (1), (3) and (9). (Fig. 3-1-3-6.) shows the growth of prisoner colis containing the pairs of plasmids (2), (4) and (10).

Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)

Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)

Fig. 3-1-3-5. Cooperating Prisoner A coli’s growth with and without an ssrA tag

Pcon_rhlR_TT_Plux_CmR (pSB6A1) + promoter less_lasI (pSB3K3)

Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + promoter less_lasI (pSB3K3)

Fig. 3-1-3-6. Defecting Prisoner A coli’s growth with and without an ssrA tag

From (Fig. 3-1-3-5.) and (Fig. 3-1-3-6.), prisoner coli with CmRssrA were not able to grow without C4HSL (pink open circle) while the prisoner coli expressing CmR with an ssrA tag grew (green open circle). We confirmed that our improved parts, Pcon_rhlR_TT_Plux_CmRssrA, had expressed CmR when induced by C4HSL.

3.2.2. 3OC12HSL-dependent CmRssrA expression (Prisoner B)

The experiment was also conducted for Prisoner B. The cell growth of the prisoner colis which contain the pairs of plasmids (5) – (8), (11), and (12) were also measured every hour for eight hours. (Fig. 3-1-3-7.) shows the growth of prisoner colis containing the pairs of plasmids (5), (7) and (11). (Fig. 3-1-3-8.) shows the growth of prisoner colis containing the pairs of plasmids (6), (8) and (12).

Pcon_lasR_TT_Plux_CmR (pSB6A1) + Plac_rhlI (pSB3K3)

Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) + Plac_rhlI (pSB3K3)

Fig. 3-1-3-7. Cooperating Prisoner B coli’s growth with and without an ssrA tag

Pcon_lasR_TT_Plux_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)

Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) + promoter less_rhlI (pSB3K3)

Fig. 3-1-3-8. Defecting Prisoner B coli’s growth with and without an ssrA tag

From (Fig. 3-1-3-7.) and (Fig. 3-1-3-8.), Prisoner coli with CmRssrA were not able to grow without 3OC12HSL (pink open circle) while the Prisoner coli expressing CmRssrA grew (green open circle). We confirmed that our improved parts, Pcon_lasR_TT_Plux_CmRssrA, had expressed CmR when induced by 3OC12HSL.

From the results above, we can say that the leaked CmR protein was degraded immediately because of the ssrA tag added to the C-terminal of the CmR protein. These results show the improved function of AHL-dependent CmR expression.

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_lasR_TT_Plux_CmRssrA (pSB6A1) + Plac_rhlI (pSB3K3)

Fig. 3-1-4-3.

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

Fig. 3-1-4-4.

　　　　　　
(5) Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)

Fig. 3-1-4-5.

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

Fig. 3-1-4-6.

(7)Negative contorol 1 : Pcon_rhlR_TT_promoter less_CmR (pSB6A1) + Plac_lasI (pSB3K3)

Fig. 3-1-4-7.

(8)Negative contorol 2 : Pcon_rhlR_TT_promoter less_CmR (pSB6A1) + promoter less_lasI (pSB3K3)

Fig. 3-1-4-8.

　　　　　　
(9) Pcon_lasR_TT_Plux_CmR (pSB6A1) + Plac_rhlI (pSB3K3)

Fig. 3-1-4-9.

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

Fig. 3-1-4-10.

　　　　　　
(11)Negative contorol 1 : Pcon_lasR_TT_promoter less_CmR (pSB6A1) + Plac_rhlI (pSB3K3)

Fig. 3-1-4-11.

　　　　　　
(12)Negative contorol 2 : Pcon_lasR_TT_promoter less_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)

Fig. 3-1-4-12.

　　　　　　
4.2. Assay Protocol

4.2.1. C4HSL-dependent CmR expression assay (Prisoner A)

-samples
Pcon_rhlR_TT_Plux_cmR (pSB6A1) ＋ Plac_lasI (pSB3K3)　
Pcon_rhlR_TT_Plux_cmR (pSB6A1) ＋ promoter less_lasI (pSB3K3)　
Pcon_rhlR_TT_promoter less_cmR (pSB6A1) ＋ Plac_lasI (pSB3K3)　
Pcon_rhlR_TT_promoter less_cmR (pSB6A1) ＋ promoter less_lasI (pSB3K3)

-Procedure
1. Prepare overnight cultures (n=2) 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 and Kan 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 containing Amp and Kan (3 mL) + 50 microM C4HSL (30 microL) + Chloramphenicol (100 microg/mL)
   ②) LB containing Amp and Kan (3 mL) + DMSO (3 microL) + Chloramphenicol (100 microg/mL)
6. Incubate the samples of cells at 37°C for more than 8 hours.
   Measure OD₅₉₀ every hour.

4.2.2. 3OC12HSL-dependent CmR expression assay (Prisoner B)

-samples
Pcon_lasR_TT_Plux_CmR (pSB6A1) + Plac_rhlI (pSB3K3)　
Pcon_lasR_TT_Plux_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)　
Pcon_lasR_TT_promoter less_CmR (pSB6A1) + Plac_rhlI (pSB3K3)　
Pcon_lasR_TT_promoter less_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)

-Procedure
1. Prepare overnight cultures (n=2) 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 and Kan 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 containing Amp and Kan (3 mL) + 5 microM 3OC12HSL (3 microL) + Chloramphenicol (100 microg/mL)
   ②) LB containing Amp and Kan (3 mL) + DMSO (3 microL) + Chloramphenicol (100 microg/mL)
6. Incubate the samples of cells at 37°C for more than 8 hours.
   Measure OD₅₉₀ every hour.

4.2.3. C4HSL-dependent CmRssrA expression assay(Prosiner A)

-samples
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) ＋ Plac_lasI (pSB3K3)　
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) ＋ promoter less_lasI (pSB3K3)　
Pcon_rhlR_TT_Plux_CmR (pSB6A1) ＋ Plac_lasI (pSB3K3)　
Pcon_rhlR_TT_Plux_CmR (pSB6A1) ＋ promoter less_lasI (pSB3K3)　
Pcon_rhlR_TT_promoter less_CmR (pSB6A1) ＋ Plac_lasI (pSB3K3)　
Pcon_rhlR_TT_promoter less_CmR (pSB6A1) ＋ promoter less_lasI (pSB3K3)

-Procedure
1. Prepare overnight cultures (n=2) 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 and Kan 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 containing Amp and Kan (3 mL) + 50 microM C4HSL (30 microL) + Chloramphenicol (100 microg/mL)
   ②) LB containing Amp and Kan (3 mL) + DMSO (30 microL) + Chloramphenicol (100 microg/mL)
6. Incubate the samples of cells at 37°C for more than 8 hours.
   Measure OD₅₉₀ every hour.

4.2.4. 3OC12HSL-dependent CmRssrA expression assay (Prisoner B)

-samples
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) + Plac_rhlI (pSB3K3)　
Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) + promoter less_rhlI (pSB3K3)　
Pcon_lasR_TT_Plux_CmR (pSB6A1) + Plac_rhlI (pSB3K3)　
Pcon_lasR_TT_Plux_CmR (pSB6A1) + promoter less_rhlI (pSB3K3)　
Pcon_lasR_TT_promoter less _CmR (pSB6A1) + Plac_rhlI (pSB3K3)　
Pcon_lasR_TT_promoter less _CmR (pSB6A1) + promoter less_rhlI (pSB3K3)

-Procedure
1. Prepare overnight cultures (n=2) 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 and Kan 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 containing Amp and Kan (3 mL) + 5 microM 3OC12HSL (3 microL) + Chloramphenicol (100 microg/mL)
   ②) LB containing Amp and Kan (3 mL) + DMSO (3 microL) + Chloramphenicol (100 microg/mL)
6. Incubate the samples of cells at 37°C for more than 8 hours.
   Measure OD₅₉₀ every hour.

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