Difference between revisions of "Team:Tokyo Tech/Toxin Assay/mazEF System Assay"
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| − | <p class="normal_text">The biggest attraction of the TA system is that it is able to control cell growth and synthesis of protein. In this experiment, <span style= | + | <p class="normal_text">The biggest attraction of the TA system is that it is able to control cell growth and synthesis of protein. In this experiment, <span style="font-style: italic;">mazE</span> expression was induced by the addition of IPTG(2 mM) after <span style="font-style: italic;">mazF</span> 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. |
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Revision as of 20:05, 18 October 2016
3-1-2 mazEF system Assay
Contents
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. 3-1-2-2-1 Plasmid diagram of E. coli C
E. coli B : carrying the PBAD-rbs -mazF-Pcon-rbs-gfp (pSB6A1) , Plac-rbs (pSB3K3)
Fig. 3-1-2-2-1 Plasmid diagram of E. coli B
E. coli D : carrying the PBAD-rbs -mazF-Pcon-rbs-gfp (pSB6A1) , Plac-rbs-mazE (pSB3K3)
Fig. 3-1-2-2-1 Plasmid diagram of E. coli D
It has been found that the expression of A is larger than that of B. 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 RFU(Relative Fluorescece Units) of GFP were measured at several time points.
1-3. Results
From the result in Fig.3-1-3-2 and Fig.3-1-3-3, 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. 3-1-2-1-3-1 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
1) E. coli C : PBAD-rbs (pSB6A1) , Plac-rbs (pSB3K3)
2) E. coli A : Pcon-rbs-gfp (pSB6A1) , Plac-rbs (pSB3K3)
3) E.coli D : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Plac-rbs-mazE (pSB3K3)
4) E. coli B : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Plac-rbs (pSB3K3)
PBAD (BBa_I0050) , Plac (BBa_R0010) , Pcon (BBa_R0040) , gfp (BBa_E0040) , rbs (BBa_B0034) , 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. 3-1-2-2-1 Plasmid diagram of E. coli C
E. coli E : carrying the PBAD-rbs-mazF-Pcon-rbs-gfp (pSB6A1) , vectoc (pSB3K3)
Fig. 3-1-2-2-1 Plasmid diagram of E. coli E
E. coli F : carrying the PBAD-rbs-mazF-Pcon-rbs-gfp (pSB6A1) , Pcon-rbs-mazE (pSB3K3)
Fig. 3-1-2-2-1 Plasmid diagram of E. coli F
E. coli G : carrying the PBAD-rbs-mazF-Pcon-rbs-gfp (pSB6A1) , Pcon-rbs(weak)-mazE (pSB3K3)
Fig. 3-1-2-2-1 Plasmid diagram of E. coli G
In order to adjust the amount of expression of mazE, two types of RBS (B0034 and J61117: J61117 is weaker than B0034) was used. Using these plasmids, we tried clarifying stoichiometric relation 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. 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. 3-1-2-2-3-2Time 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
We found that MazF inhibited cell growth and translation even when there was MazE.
In addition,these results suggested that there was a 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 F : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Pcon-rbs(BBa_B0034)-mazE (pSB3K3)
E. coli G : PBAD-rbs-mazF-tt-Pcon-rbs-gfp (pSB6A1) , Pcon-rbs(BBa_J61117)-mazE (pSB3K3)
PBAD (BBa_I0050) , Plac (BBa_R0010) , Pcon (BBa_R0040) , gfp (BBa_E0040) , rbs (BBa_B0034) , mazF (BBa_K1096002) , mazF (BBa_K1096001),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.
