Team:Tokyo Tech/Toxin Assay/mazEF System Assay

3-1-2 mazEF system Assay

3-1-2-1 Stop & Go

1-1. Introduction

The most attractive feature of the mazEF system is that cytotoxity of a toxin protein (MazF) is determined by the stoichiometric ratio of a toxin protein to a corresponding antitoxin protein (MazE) in cells. The story of Snow White involves sleep and resuscitate from sleep, and thus, we think that this feature is useful for our project.

1-2. Summary of the experiment

Transformants shown below were 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 is well known that the expression from Plac is usually stronger than that from PBAD. Therefore, we expected that the expression level of mazE would be finally higher than 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 Fluorescence Units (RFU) of GFP were measured at several time points.

1-3. Results

As shown in Fig. 3-1-2-1-3-1, when the mazE expression was induced 2 h after the mazF expression, cell growth resumed gradually. Similarly, the RFU of GFP also increased 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). 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. The reason is unclear, but the prior expression of mazF might damage the cells and it might take time to repair. Alternatively, the mazF expression might lead cells to the dormant state from which cells can hardly escape. We assume that this feature is advantageous for our Snow White project, because we can prolong the sleeping time of Snow White and take sufficient time for Prince to find Snow White. As a conclusion, the “Stop and Go” experiment was successful.



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

　

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 3-1-2-1, a toxin inhibits cell growth, and an antitoxin resuscitates it. However, what will happen when a toxin is expressed after the constitutive expression of antitoxin? Therefore, in the experiments of this section, we conducted a reciprocal experiment and named this experiment "Go & Stop".

2-2. Summary of the experiment

Transformants shown below were 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 expression level of mazE, two types of RBS (BBa_B0034 and BBa_J61117: J61117 is weaker than B0034) were used. Using these plasmids, we analyzed relationship between the expression ratio of mazE to mazF and overall translation efficiency in cells. 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 Fluorescence Units (RFU) of GFP were measured at several time points.

2-3. Results

Turbidity 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), indicating that cellular mazE amount over MazF amount affects cytotoxity of MazF. In other words, the cytotoxity of MazF is determined stoichiometrically. Similarly, final RFU of E. coli G was lower than that of E. coli F.
From the results of Experiment 1.2.1. and Experiment 1.2.2., it is expected that mazEF can repeatedly control cell growth.

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

(Queen's Caprice)

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. It seemed like that dormancy of Snow White was controled by Queen, we named this experiment "Queen's caprice".

3-2. 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). We believe that the repeated control of cell growth is very useful for the future biotechnology; read the Human Pracrices 3E section for details.


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