Team:Tokyo Tech/Toxin Assay/Overview
3-1-0. Overview
Contents
3-1-0. TA system
TA systems have function of controlling cell growth and are present on the genomic DNA of many microorganisms as well as on plasmid DNAs. We thought that exploiting the ability of TA systems was the best way to trace the story of Snow White, because the story includes death and recovery from death.
Preceding iGEM teams have studied the following TA systems: ccdB-ccdA (iGEM 2014 team ULB-Brussels and iGEM 2013 team UGent), Kid-Kis, Zeta-Epsilon(iGEM 2014 team Wageningen UR) and Hok-Soc(iGEM 2015 team UMaryland). The iGEM 2016 team Tokyo_Tech studied the mazEF system, which is one of the most vitally studied TA systems.
MazF (Toxin) specifically recognizes and degrades ACA sequences of mRNAs, thereby inhibiting translation and cell growth. MazE (Antitoxin for MazF) forms a MazF-MazE heterohexamer and neutralize the toxicity of MazF. If this system is used appropriately, it is possible to manipulate growth of E. coli cells as we expect, and wide range of biotechnological application is possible.
Fig. 3-1-0-1. Table. Achievement of TA System in the preceding iGEM
3-1-1. Adjustment of MazF expression
MazF has very strong ability to inhibit cell growth. A previous study have shown that such inhibition can not be restored even when MazE antitoxin is highly expressed simultaniouly2). Therefore, as a first experiment, mazF gene was placed under the downstream of PBAD, and optimal inducer (L-arabinose) concentration was examined.
Fig. 3-1-0-2.
From the result of the experiment 1.1., the optimal arabinose concentration was found to be 0.02%. However, under arabinose concentration of 0.2%, GFP fluorescence intensity decreased markedly; we were unable to explain why such decrease occurred, but the same results were obtained repeatedly. Anyway, 0.02% of arabinose did not decrease GFP fluorescence, and thus, this concentration was used for further studies.
3-1-2. mazEF System Assay
3-1-2-1. Stop & Go
Based on the above result, we conducted an experiment where cell growth was controlled by using the mazEF system.
In the first experiment, MazF was first expressed and MazE was expressed later to see if cell growth can be recovered by the neutralizing activity of MazE. This experiment was named "Stop & Go".
Fig. 3-1-0-3.
In the experiment 1.2.1., MazE expression was induced 2 h after MazE expression. It turned out that cell growth was inhibited immediately after MazF expression and recovered by MazE.
The revival seemed to take longer time than expected, suggesting that MazF activity might damage cells seriously. We would like to emphasize that the result was different from that in the earlier study mentioned above and postulate that expression level, duration, timing of MazF may affect the result. From this result, we concluded that "Stop & Go" experiment was successful and the system can be used for reproducing the important scene of Snow White.
3-1-2-2. Go & Stop
In the following experiment, a reciprocal experiment to the "Stop & Go" experiment was performed; it was named "Go & Stop" experiment. the antitoxin was expressed from the beginning of the culture (= constitutively expressed) and toxin expression was induced at the middle of the culture.
From the results of the Exp.1.2.2, it was found that the higher the expression level of MazE was, the larger the number of E. coli cells was observed in the stationary phase.
Furthermore, there was a correlation between translation activity in the cells (judged from GFP expression) and expression level of MazE.
3-1-3. Control of Cell Growth
In order to further confirm desirable control of cell growth, we conducted an assay using an LB agar plate containing an inducer for toxin or antitoxin.
This experiment was named “TA system ~Queen’s caprice～”, because Queen controls 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).（保留わかんない）
However, E. coli cells expressing antitoxin initially were unable to control colonization.（保留わかんない）
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 E. 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.
