Team:Tokyo Tech/Toxin Assay/Queens capricious

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3-1-4 YafO System Assay

Contents

1. Introduction

2. Summary of the Experiment

   2-1. Confirming YafO Function as Toxin on Agar plates

   2-2. Toxin-Antitoxin Assay

3. Results

   3-1. Confirming YafO Function as Toxin on Agar plates

   3-2. Toxin-Antitoxin Assay

4. Discussion

5. Materials and Methods

   5-1. Strain

   5-2. Plasmid

     5-2-1. Assay to Confirm YafO function as toxin on agar plates

     5-2-2. toxin-antitoxin assay

   5-3. Assay Protocol

     5-3-1. Confirming YafO function as toxin on agar plate

     5-3-2. toxin-antitoxin assay

6. Reference

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.