Team:Tokyo Tech/Experiment/ssrA tag degradation assay
ssrA tag degradation assay
contents
1. Introduction
2. Summary of the Experiment
2.1. AHL-dependent CmR expression
2.1.1 C4HSL-dependent CmR expression (Prisoner A)
2.1.2 3OC12HSL-dependent CmR expression (Prisoner B)
2.2. Insertion of an ssrA degradation tag to CmR
3. Results
3.1. AHL-dependent CmR expression
3.2. 3OC12HSL-dependent CmR expression (Prisoner B)
4. Materials and Methods
4.1. Construction
4.2. Assay Protocol
4.2.1. C4HSL-dependent CmR expression assay ([Cm] = 75 microg/mL)
4.2.2. 3OC12HSL concentration-dependent CmR expression assay
5. Reference
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 negative control pairs of plasmids for (1), and (2), respectively.
|
|
Fig. 3-1-2-2. Pair 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 negative control pairs of plasmids for (5), and (6), respectively.
|
|
Fig. 3-1-2-4. Pair of plasmids for the experiment of 3OC12HSL-dependent CmR expression |
2.2. Insertion of an ssrA degradation tag to CmR
At the first stage of wet experiment, Prisoner colis containing the pairs of plasmids (1) and (2), which were the previously existing circuits 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) in Prisoner A coli (Fig. 3-1-2-2.). Our modeling result shows that the influence of the leakage was not reduced by increasing the chloramphenicol (Cm) concentration, which was one of our solutions. Please refer here.
For precise implementation of our payoff matrix, suggestions from modeling (link to modeling) allowed us to successfully solve the influence of the leakage by adding an ssrA degradation tag right after the CmR gene. ((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. Pair of plasmids for the ssrA degradation tag assay of Prisoner A | |
|
|
Fig. 3-1-2-8. Pair 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.
Sample : Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)
|
|
Fig. 3-1-3-1. Cooperating Prisoner A coli’s growth with chloramphenicol |
Fig. 3-1-3-2. Defecting Prisoner A coli’s growth with chloramphenicol |
Fig. 3-1-2-7. Pair of plasmids for the ssrA degradation tag assay of Prisoner A |
Fig. 3-1-3-1. Payoff matrix of Prisoner A coli with 75 microg/mL Cm |
3.2. Replicating the Payoff Matrix of Prisoner B coli
The cell growth of the Prisoner colis containing the pairs of plasmids (3) and (4) in the lower chloramphenicol (Cm) concentration (75 microg/mL) and in the lower 3OC12HSL concentration were measured. The payoff matrix was realized as the following.
|
|
Fig. 3-1-3-2. Payoff matrix of Prisoner B coli with gfp in 75 microg/mL Cm and 1 nM 3OC12HSL |
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) + Pcon_gfp_rhlI (pSB3K3)
|
|
Fig. 3-1-4-3. |
(4) Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) + promoter less_rhlI (pSB3K3)
|
|
Fig. 3-1-4-4. |
4.2. Assay Protocol
4.2.1. C4HSL-dependent CmR expression assay ([Cm] = 75 microg/mL)
-samples
(1) Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) ＋ Plac_lasI (pSB3K3)
(2) Pcon_rhlR_TT_Plux_CmRssrA (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 (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.)
4.2.2. 3OC12HSL concentration-dependent CmR expression assay
-samples
(3) Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) ＋ Pcon_gfp_rhlI (pSB3K3)
(4) Pcon_lasR_TT_Plux_CmRssrA (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 (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.
a) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 1 microM 3OC12HSL (3 microL) + Chloramphenicol (75 microg/mL)
b) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (75 microg/mL)
6. Grow the samples of cells at 37°C for more than 8 hours.
Measure optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)
5. Reference
1. Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619