Team:Tokyo Tech/Toxin Assay/Queens capricious
3-1-2 mazEF system Assay
Contents
1. Introduction
Our project, the story of “Snow White” is constructed based on mazEF system, which is one of toxin-antitoxin (TA) system on E. coli genomic DNA. At the same time, we are interested in other TA systems and we carried out assay using yafNO system.
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.
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)
Fig. 3-1-2-1-5-1
2) GFP : Pcon-rbs-gfp (pSB6A1) , Plac-rbs (pSB3K3)
Fig. 3-1-2-1-5-2
3) MazF , MazE : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Plac-rbs-mazE (pSB3K3)
Fig. 3-1-2-1-5-3
4) MazF : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Plac-rbs (pSB3K3)
Fig. 3-1-2-1-5-4
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.
2-3. Results
E. coli encoded mazE which is on downstream of weak RBS(J61117) has more turbidity than E. coli encoded mazE which is on downstream of normal RBS(B0034). Both of those E. coli would reach stationary phase when there is little RFU of GFP. E. coli encoded mazE which is on downstream of normal RBS (B0034) reached almost the same stationary phase as E. coli without TA system
Fig. 3-1-2-2-3-1.Time vs Turbidity (Go &Stop)
Fig. 3-1-2-2-3-2Time 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. 3-1-2-2-3-3translation efficiency of each E. coli.
2-4. Discussion
From this experimental result, we found that MazF inhibits cell growth and translation even when there is MazE. Additionally, Fig 3-1-2-2-3-3 showed that the more MazE was expressed, the higher the translation efficiency got. Therefore, the result suggests that MazF inhibits translation, and MazE resuscitates the translation inhibition; the resuscitation correlates MazE expression. This experimental result is associated with anamnestic experimental results which indicated that MazE and MazF form a hexamer (MazF2-MazE2-MazF2). The results of Experiment1.2.1. and Experiment1.2.2. 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) Vector : Pbad-rbs(pSB6A1) , Plac-rbs (pSB3K3)
Fig. 3-1-2-2-5-1.
2) GFP : Pcon-rbs-gfp (pSB6A1) , Plac-rbs(pSB3K3)
Fig. 3-1-2-2-5-2.
3) MazF , MazE(weak) : Pbad-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Pcon-rbs(weak)-mazE (pSB3K3)
Fig. 3-1-2-2-5-3.
4) MazF + MazE : Pbad-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Pcon-rbs-mazE(pSB3K3)
Fig. 3-1-2-2-5-4.
5) MazF : Pbad-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , vector (pSB3K3)
Fig. 3-1-2-2-5-5.
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.
