Team:Tokyo Tech/Toxin Assay/Overview
3-1-0. Overview
Contents
3-1-0. TA system
TA system has a function of controlling the cell growth present on the genomic DNA of many microorganisms.
Preceding iGEM teams studied on the following TA systems: ccdB-ccdA (ULB-Brussels 2014 and UGent 2013), Kid-Kis, Zeta-Epsilon(Wageningen UR 2014) and Hok-Soc(UMaryland 2015).
In iGEM2016 Tokyo_Tech team, we studied on mazEF system, which is actively studied as the TA system.
MazF (Toxin) is specifically recognize and degrade the ACA sequence of mRNA, inhibiting the translation and cell growth. MazE(Antitoxin for MazF) form a MazF-MazE heterohexamer complex, it has a function to neutralize the toxicity of MazF. If you can use it well, it is possible to manipulate growth of E. coli in freely, a wide range of applications is expected.
We conducted the following experiment in order to pave the application of further TA system.
Fig. 3-1-0-1. Table. Achievement of TA System in the preceding iGEM
MazF (Toxin) specifically recognizes and degrades the ACA sequence of mRNA, inhibiting the translation and cell growth. MazE(Antitoxin for MazF) forms a MazF-MazE heterohexamer complex, which has a function that neutralizes the toxicity of MazF1). If you can use the system well, it is possible to regulate growth of E. coli freely, and wide range of applications are expected.
We conducted the following experiment to pave the application of further TA system.
3-1-1. Adjustment of MazF expression
MazF is very strong ability to inhibit cell growth, it has been reported not restored even to express too expressed antitoxin2). Therefore, to clarify the expression of MazF capable of suppressing growth of E. coliv , we examined optimal inducer concentration, downstream from the PBAD.
Fig. 3-1-0-2.
From the result of the experiment 1.1., when the assay of TA system using genetic circuit above, the optimal arabinose concentration was found to be 0.02%. However, when Arabinose concentration of 0.2%, GFP fluorescence intensity falls markedly.
3-1-2. mazEF System Assay
3-1-2-1. Stop & Go
Concentration of arabinose to express the appropriate amount of MazF was confirmed in the previous experiment. Based on the result, we have conducted an experiment whether it is control of growth by using the mazEF system.
We named this experiment as "Stop & Go" because it was to resume growth from suppression the cell growth.
Fig. 3-1-0-3.
From the results of the experiment 1.2.1., MazE was induced 2 h after MazE expression, and about 8 h later, cell growth that had stopped was recovered .
Although the revival took longer than expected, suggesting the strength of MazF as the toxin from this result.
3-1-2-2. Go & Stop
In Experiment 1.2.1, it has been found that growth is inhibited by toxin and revived by antitoxin.
However, if to express the toxin after expression of antitoxin, we wonder how to behave is growth?
So if mazF expressed after MazE constitutively expressed, we studied how to grow.
From the results of the Exp.1.2.2, it was found that the larger expression of A is, the larger the number of E. coli is in the Stationary Phase.
Further, it was found that there is a correlation efficiency of translation and expression of MazE.
3-1-3. Control of Cell Growth
In order to further secure the control of cell growth, we conducted assay of TA system on the LB agar plate containing inducer of toxin or antitoxin.
This experiment was named “TA system ~Queen’s capricious～”, because it seemed that princess control a sleep for snow white,
From the results of the Experiment 1.3., it has been found that A cultured in a medium containing inducer of toxin initially may be able to control the colonization by the presence or absence of IPTG(inducer of antitoxin).
But E. coli cultured on a medium containing inducer of antitoxin initially were unable to control the colonization.
This result might indicate that function of toxin is not only growth inhibition
If all the above mentioned results are mobilized, it may become possible to freely control of the growth, make of the story with Escherichia coli, product the material production in any timing.
3-1-4. Reference
1) Yonglong Zhang, Junjie Zhang, Klaus P. Hoeflich, Mitsuhiko Ikura, Guoliang Qing, and Masayori Inouye, MazF Cleaves Cellular mRNAs Specifically at ACA to Block Protein Synthesis in Escherichia coli. Molecular Cell, Vol. 12, 913–923.
2) Hazan, R., B. Sat, and H. Engelberg-Kulka. Escherichia coli mazEFmediated cell death is triggered by various stressful conditions. J. Bacteriol.186:3663–3669.
