Team:Tokyo Tech/Toxin Assay/Queens capricious
3-1-4 YafO 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.
2. Summary of the Experiment
2-1. Confirming YafO Function as Toxin on Agar plates
We first confirmed YafO function by observing formation of colonies on agar plates. This experiment was carried out using E. coli which can induce YafO expression by arabinose and E. coli without yafO gene. Construction of plasmids used in this experiment is shown in Fig. 2-1-1. We inoculated four types of E. coli differently on agar plates with or without arabinose.
Fig. 2-1-1.
2-2. Toxin-Antitoxin Assay
From previous experiment, we confirmed that YafO works as toxin. Next, we confirmed whether the cell growth recovers with antitoxin YafN after inhibition from YafO. Construction of plasmids used in this experiment is shown as Fig. 2-2-1. We prepared E. coli which can induce YafO expression by arabinose and YafN expression by lactose. As comparisons, we also carried out same experiment with E. coli containing no yafO gene, no yafN gene, or none of them. These E. coli were respectively cultured in media with arabinose in order to express YafO, then IPTG were added to the cultures in order to express YafN. We confirmed YafN function against YafO by measuring turbidity and RFU (relative fluorescence units) of GFP.
Fig. 2-2-1.
3. Results
3-1. Confirming YafO Function as Toxin on Agar plates
Four types of E. coli shown in Fig. 2-1-1 were inoculated on agar plates with or without 0.2% arabinose, and incubated at 37°C. As a result, E. coli containing plasmid (a) and one containing plasmid (c) didn’t form any colonies, although all types of E. coli formed colonies on agar plate containing no arabinose (A). From this result, cell growth was inhibited by inducing expression of YafO.
Particularly, like E. coli containing plasmid (d), E. coli containing plasmid (c) formed fluorescent colonies on agar plate (A), and like E. coli containing plasmid (a), didn’t form any colonies on agar plate (B). These results insist that genes on plasmid (c) were working for sure.
Fig. 3-1-1.
3-2. toxin-antitoxin assay
Four types of E. coli (shown as Fig. 2-1-1) were inoculated in liquid media, respectively. When turbidity of culture reached 0.03, arabinose was added (final concentration 0.02%) to each culture. After two hours incubation with arabinose, IPTG was also added (final concentration 2 mM). Time-dependent change of RFU and turbidity is shown as Fig. 3-2-1. The graph (A) shows that, even though E. coli containing plasmids (a) has yafN gene, it couldn’t make the cell growth recover, like one containing plasmids (c) (lack yafN gene). Also, from graph (B), no recovery of RFU was shown on E coli containing plasmid (a), and its time-dependent change of RFU was similar to that of turbidity.
Fig. 3-2-1.
