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

A pSB6A1-based plasmid containing both the PBAD (BBa_I0050)-rbs (BBa_B0034)-mazF (BBa_K1096002) and the Pcon (BBa_R0040)-rbs (BBa_B0034)-gfp (BBa_E0040) cassettes 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 in which growth was controlled by the TA system. Samples were incubated with vigorous shaking at 37℃. When turbidity turned to be 0.03, we added arabinose of which concentration is 0.02% (to induce MazF expression). 2 h after the addition of arabinose, we added IPTG so that the final concentration becomes 2 mM (to induce MazE expression). And we measured the turbidity and RFU of GFP at proper time.



Fig. 3-1-2-1-2-1.



Fig. 3-1-2-1-2-2.



Fig. 3-1-2-1-2-3.


1-3. Results

It was found from fig.3-1-3-2 and fig.3-1-3-3 that MazF inhibited cell growth. MazE was induced 2 h after MazE expression, and about 8 h later, cell growth was recovered that had stopped. From these results, it was suggested that E. coli whose cell growth was inhibited by MazF was able to resuscitate by expression of MazE.




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

　From these results, it was found that using TA system can resuscitate the inhibited cell growth. It took much time to resuscitate cell growth. It can be attributed to inhibition of protein synthesis by MazF and the formation of MazE-MazF complex. It is necessary for MazE to be combined with MazF so that MazE acts as an antitoxin of MazF. In addition, it is expected that the production speed of MazE declined because of the translation inhibition caused by MazF.

5. Materials and Methods

5-1. Construction

-Strain All the samples were XL1-Blue strain.
-Plasmids
1) promoter only : PBAD-rbs (pSB6A1) , Plac-rbs (pSB3K3)
2) GFP : Pcon-rbs-gfp (pSB6A1) , Plac-rbs (pSB3K3)
3) MazF , MazE : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Plac-rbs-mazE (pSB3K3)
4) MazF : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Plac-rbs (pSB3K3)





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

A pSB6A1-based plasmid containing both the PBAD (BBa_I0050)-rbs (BBa_B0034)-mazF (BBa_K1096002) and the Pcon (BBa_R0040)-rbs (BBa_B0034)-gfp (BBa_E0040) cassettes was constructed. Furthermore, a pSB3K3-based plasmid containing the Pcon (BBa_R0010)-rbs(BBa_B0034)-mazE(BBa_K1096001) cassette was constructed. These plasmids were co-introduced into E. coli. In addition, a pSB3K3-based plasmid containing the Pcon-rbs(BBa_J61117)-mazE cassette was constructed, using RBS (BBa_J61117), which is weaker than RBS (BBa_B0034). Using these plasmids, we tried clarifying stoichiometric proportion by changing the expression of MazE with two types of RBS (B0034 and J61117) downstream of Pcon.



Fig. 3-1-2-2-2-1.



Fig. 3-1-2-2-2-2.



Fig. 3-1-2-2-2-3.



Fig. 3-1-2-2-2-4.



Fig. 3-1-2-2-2-5.

2-3. Results

E. coli encoded MazE downstream of weak RBS (J61117) reached stationary phase with smaller turbidity and RFU of GFP than that of normal RBS (B0034). E. coli encoded mazE downstream of normal RBS (B0034) reached almost the same stationary phase as E. coli without TA system.

Fig. 3-1-2-2-3-1.


Fig. 3-1-2-2-3-2


Fig. 3-1-2-2-3-3


Fig. 3-1-2-2-3-4


2-4. Discussion

From this experimental result, we found that MazF inhibits cell growth and translation with MazE. Additionally, Fig 3.1.2.2.8 showed that more expression level of MazE, less likely inhibited translation of MazF stoichiometrically.
Therefore the result suggests that MazF inhibits translation, and mazE resuscitates the translation inhibition; resuscitation correlates MazE expression level. This experimental result is associated with anamnestic experimental results indicated that MazE and MazF form a hexamer (MazF2-MazE2-MazF2)
The results of Experiment 1.2.1. and Experiment 1.2.2. seem that using mazEF System can repeat the inhibition of cell growth and translation by a toxin, and resuscitation of cell growth and translation by an antitoxin.

2-5. Materials and Methods

2-5-1. Construction

-Strain
All the samples were XL1-Blue strain.

-plasmid
1) Pbad _rbs(pSB6A1) + Plac_rbs (pSB3K3)
2) Pcon_rbs_gfp (pSB6A1) + Plac_rbs(pSB3K3)
3) Pbad_rbs_mazF_TT_Pcon_rbs_gfp (pSB6A1) + Pcon_rbs(weak)_mazE (pSB3K3)
4) Pbad_rbs_mazF_TT_Pcon_rbs_gfp (pSB6A1) + Pcon_rbs_mazE (pSB3K3)
5) Pbad_rbs_mazF_TT_Pcon_rbs_gfp (pSB6A1) + vector only (pSB3K3)





2-5-2. Assay Protocol

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