Team:Tokyo Tech/Toxin Assay/mazEF System Assay

3-1-2 mazEF system Assay

3-1-2-1 Stop & Go

1-1. Introduction

The biggest attraction of the TA system is that it is able to control cell growth and synthesis of protein. In this experiment, mazE expression was induced by the addition of IPTG (2 mM) after mazF expression was induced by the addition of arabinose (0.02%). As a result, it was able to resuscitate from a state of being inhibited cell growth. We named this experiment as "Stop & Go" because it was to resuscitate growth from inhibiting cell growth.

1-2. Summary of the Experiment

Transformants as shown below was prepared.

E. coli A: carrying the Pcon ‐ rbs ‐ gfp (pSB6A1) , Plac ‐ rbs (pSB3K3)


Fig. 3-1-2-2-1 Plasmid diagram of E. coli A




E. coli C: carrying the PBAD ‐ rbs (pSB6A1) , Plac ‐ rbs (pSB3K3)


Fig. 3-1-2-2-2 Plasmid diagram of E. coli C




E. coli B: carrying the PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) , Plac ‐ rbs (pSB3K3)


Fig. 3-1-2-2-3 Plasmid diagram of E. coli B




E. coli D: carrying the PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) , Plac ‐ rbs ‐ mazE (pSB3K3)


Fig. 3-1-2-2-4 Plasmid diagram of E. coli D



It has been found that the expression of Plac is larger than that of PBAD. Therefore, we expected that the expression of mazE would be finally larger than the that of mazF. Samples were incubated with vigorous shaking at 37℃. When turbidity reached 0.03, 0.02% arabinose was added to induce mazF expression. Two hours after the addition of arabinose, 2 mM IPTG was added to induce mazE expression. The turbidity and Relative Fluorescece Units (RFU) of GFP were measured at several time points.

1-3. Results

From the result in Fig. 3-1-2-1-3-1, it was found that MazF inhibited cell growth. The mazE expression was induced 2 h after the mazF expression, and about 8 h later, cell growth resumed. Similarly, the RFU of GFP was also resumed(Fig. 3-1-2-1-3-2).



Fig. 3-1-2-1-3-1 Time vs Turbidity (Stop & Go)



Fig. 3-1-2-1-3-2 Time vs RFU of GFP (Stop & Go)


4. Discussion

　Before starting the experiments, we anticipated that recovery from the growth inhibition by MazF would be observed immediately after the induction of mazE expression. However, it took 8 h for us to observe the recovery.

5. Materials and Methods

5-1. Construction

-Strain All the samples were XL1 ‐ Blue strain.
-Plasmids
   E. coli A: Pcon ‐ rbs ‐ gfp (pSB6A1) , Plac ‐ rbs (pSB3K3)

E. coli C: PBAD ‐ rbs (pSB6A1) , Plac ‐ rbs (pSB3K3)

E. coli B: PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) , Plac ‐ rbs (pSB3K3)

E. coli D: PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) , Plac ‐ rbs ‐ mazE (pSB3K3)

PBAD (BBa_I0500) , Plac (BBa_R0010) , Pcon (BBa_R0040) , gfp (BBa_E0040) , rbs (BBa_B0034) , mazE (BBa_K1096001) , mazF (BBa_K1096002) , tt (BBa_B0015)





5-2. Assay Protocol

Pre-culture
1. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).

2. Incubate with vigorous shaking for 12 h.


　　 Incubation and Assay
1. Measure the turbidity of the pre-cultures.

2. Dilute the pre- cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin.

3. Incubate with vigorous shaking so that turbidity becomes 0.03.

4. Add arabinose so that the final concentration becomes 0.02%.

5. 2 h after the addition of arabinose, we added IPTG so that the final concentration becomes 2 mM.

6. Incubate with vigorous shaking for 24 h, and measure turbidity and RFU of GFP at the proper time.

3-1-2-1 Go & Stop

2-1. Introduction

In Experiment 1-2-1, we found that a toxin inhibits cell growth, and an antitoxin resuscitates it. However, what will happen when a toxin is expressed after the antitoxin constitutive expression? Therefore, we conducted the experiment. Since cell growth was resuscitated after cells had grown, we named this experiment, "Go & Stop".

2-2. Summary of the Experiment

Transformants as shown below was prepared. E. coli A: carrying the Pcon - rbs - gfp (pSB6A1) , Plac - rbs (pSB3K3)


Fig. 3-1-2-2-1 Plasmid diagram of E. coli A




E. coli C: carrying the PBAD - rbs (pSB6A1) , Plac - rbs (pSB3K3)


Fig. 3-1-2-2-2 Plasmid diagram of E. coli C




E. coli E: carrying the PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) , vector (pSB3K3)


Fig. 3-1-2-2-3 Plasmid diagram of E. coli: E




E. coli F: carrying the PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) , Pcon ‐ rbs ‐ mazE (pSB3K3)


Fig. 3-1-2-2-4 Plasmid diagram of E. coli F



E. coli G: carrying the PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) , Pcon ‐ rbs(weak) ‐ mazE (pSB3K3)


Fig. 3-1-2-2-5 Plasmid diagram of E. coli G



In order to adjust the amount of expression of mazE, two types of RBS (BBa_B0034 and BBa_J61117: J61117 is weaker than B0034) was used. Using these plasmids, we tried clarifying stoichiometric relationship between A and B by changing the expression of mazE. Samples were incubated with vigorous shaking at 37℃. When turbidity reached 0.03, 0.02% arabinose was added to induce mazF expression. The turbidity and Relative Fluorescece Units (RFU) of GFP were measured at several time points.

2-3. Results

Increase in turbidity and RFU of E. coli G stopped earlier than that of E. coli F(Fig. 3-1-2-2-3-1 and Fig. 3-1-2-2-3-2). As a result, turbidity and the RFU of E. coli F in the stationary phase was smaller than that of E. coli G.

Fig. 3-1-2-2-3-1 Time vs Turbidity (Go &Stop)


Fig. 3-1-2-2-3-2 Time vs RFU of GFP (Go &Stop)


Calculation of the change of RFU of GFP / Turbidity per unit time (translation efficiency) indicates that the expression level of mazE correlated with the translation efficiency(Fig 3-1-2-2-3-3).

Fig. 3-1-2-2-3-3 Translation efficiency of each E. coli.


2-4. Discussion

We found that MazF inhibited cell growth and translation even when there was MazE. In addition,these results suggested that there exsited relation between the resuscitation and mazE expression.

   From results of Experiment1.2.1. and Experiment1.2.2., it was expected that mazEF can repeatedly control cell growth.

2-5. Materials and Methods

2-5-1. Construction

-Strain
All the samples were XL1 ‐ Blue strain.

-plasmid
   E. coli C: PBAD ‐ rbs (pSB6A1) , Plac ‐ rbs (pSB3K3)

E. coli A: Pcon ‐ rbs ‐ gfp (pSB6A1) , Plac ‐ rbs (pSB3K3)

E.coli E: PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) , vector (pSB3K3)

E. coli G: PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) , Pcon ‐ rbs(BBa_J61117) ‐ mazE (pSB3K3)

E. coli F: PBAD ‐ rbs ‐ mazF ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1) , Pcon ‐ rbs(BBa_B0034) ‐ mazE (pSB3K3)

PBAD (BBa_I0500) , Plac (BBa_R0010) , Pcon (BBa_R0040) , gfp (BBa_E0040) , rbs (BBa_B0034) , mazE (BBa_K1096001) , mazF (BBa_K1096002) , tt (BBa_B0015) , weak rbs (BBa_J61117)





2-5-2. Assay Protocol

Pre-culture
1. Suspend colonies on a master plate into LB medium containing ampicillin (50 microg / mL) and kanamycin (50 microg / mL).
2. Incubate with vigorous shaking for 12 h.

Incubation and Assay
1. Measure the turbidity of the pre-cultures.
2. Dilute the pre- cultures to 1 / 30 into LB medium containing 4 mL ampicillin and kanamycin.
3. Incubate with vigorous shaking so that turbidity becomes 0.03
4. Add arabinose so that the final concentration becomes 0.02%.
5. Incubate with vigorous shaking for 24 h, and measure turbidity and RFU of GFP at proper times.

3-1-2-3 mazEF System Assay on the LB Agar Plate

3-1. Introduction

　The control of cell growth by the mazEF system has been shown until the previous sections. In this section, we analyzed whether the “stop & go” experiment can be repeated many times.

2-2. Summary of the Experiment

A pSB6A1-based plasmid containing both the PBAD(BBa_I0050) - rbs(BBa_B0034) - mazF (BBa_K1096002) cassette was constructed. Furthermore, a pSB3K3-based plasmid containing the Plac(BBa_R0010) - rbs(BBa_B0034) - mazE (BBa_K1096001) cassette was constructed. These plasmids were co-introduced into E. coli. We prepared LB medium containing arabinose (Ara(+)), IPTG (IPTG (+)), both inducers (Ara(+), IPTG(+)), and no inducers (Ara (-), IPTG (-)). We tested whether the E. coli cells formed colonies on above plates by controlling the mazEF system. The procedures are shown below.

(i) Day1: Each transformant was streaked onto the Ara(+)-, IPTG(+)-, and
                  Ara(-)_IPTG(-)-plates.

(ii) Day2: The colonies on the above plates were re-streaked onto the Ara(+)_IPTG(+)-plate.                   Even when no colonies were found on the plates, the streaked areas on day 1 were
                  scratched by toothpicks and re-streaked.

(iii) Day3: The same procedure was conducted as day 2 except that Ara(+)-plate was used.

(iv) Day4: The same procedure was conducted as day 2.


Fig. 3-2-1-3-1. Overview of the experiment

3-3. Results & Discussion

From the result, it was clarified that growth of E. coli cells was repeatedly controlled by expression of mazE(Fig. 3-1-2-3-3-1 and Table 3-1-2-3-3-1). The results of this experiment are very useful in our project, and it is expected to lead to new biotechnological application.


Fig. 3-1-2-3-3-1 Control of colonization by mazEF system on the LB Agar Plate


Table 3-1-2-3-3-1 Control of colonization by mazEF system on the LB Agar Plate


3-5. Materials and Methods

3-5-1. Construction

-Strain

All the samples were XL1 ‐ Blue strain.

-Plasmids

E. coli D: PBAD ‐ rbs ‐ mazF (pSB6A1) ，Plac ‐ rbs ‐ mazE (pSB3K3)

E. coli H: PBAD ‐ rbs ‐ mazF (pSB6A1) ，Plac ‐ rbs (pSB3K3)

E. coli I: PBAD ‐ rbs (pSB6A1)　，Plac ‐ rbs (pSB3K3)

PBAD (BBa_I0500) , Plac (BBa_R0010) , rbs (BBa_B0034) , mazE (BBa_K1096001) , mazF (BBa_K1096002)





3-5-2. Assay Protocol

1) Making LB agar medium containing arabinose and IPTG.

2) E. coli are applied at 3 agar medium (in arabinose, in IPTG, in arabinose and IPTG)

3) Overnight culture at 37°C for 24 h

4) To confirm TA system, inoculate colonies of E. coli having plasmidⅠ, Ⅱ, Ⅴ at agar medium containig arabinose and IPTG

5) Overnight culture at 37°C for 24 h

6) Inoculate colonies of E. coli into agar medium containing arabinose.

7) Overnight culture at 37°C for 24 h

8) Inoculate colonies of E. coli into agar medium in arabinose and IPTG