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PBAD-rbs-yafO

Toxin-Antitoxin (TA) systems consist of stable toxin protein which attacks essential factors needed for cell growth, and unstable antitoxin protein which negates the function of toxin. In E. coli genome, there are many operons coding gene of these proteins. Among them, toxin proteins which work as RNA interferase cleaving unspecified mRNA are particularly known. When they function, inhibition of cell growth or cell killing can be induced. YafO is one of these toxins and YafN is co-expressed cognate antitoxin.

YafO is mRNA interferase, that is, it cleaves mRNA by endonuclease activity and inhibits protein synthesis. It is thought that YafO endonuclease activity is induced by binding to 50S subunit in 70S ribosome. Yafo is a rebosome-dependent mRNA interferase and cleaves coding regions of mRNAs.

It is also considered that YafO inhibits translation in two steps. Initially, the binding of the toxin to 70S ribosomes inhibits translation reversibly via binding with its cognate antitoxin. However, in the second step, as the toxin binding to ribosomes is prolonged, the latent ribonuclease activity of the toxin is induced to cleave mRNAs, which results in irreversible inhibition of protein synthesis.

Characterization

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.

1. Confirming YafO Function as Toxin on Agar Plates

Four types of E. coli shown in Fig. 1 were inoculated on agar medium with and without 0.2% arabinose, and incubated at 37°C. The result is shown in Fig. 3. In this figure, (A) shows agar medium without arabinose and (B) shows agar medium with 0.2% arabinose. E. coli containing plasmid (a) and one containing plasmid (c) couldn’t form any colonies on agar medium (B), while all types of E. coli was able to form colonies on agar medium (A). From this result, cell growth was inhibited by inducing expression of YafO.

Particularly, E. coli containing plasmid (c) formed fluorescent colonies as E. coli containing plasmid (d) on agar medium (A), and couldn’t form any colonies as E. coli containing plasmid (a) on agar medium (B). This result insists that genes on plasmid (c) are working for sure.

Fig.3 Confirming YafO function as toxin on agar medium

Each E. coli containing (a) PBAD_rbs_yafO (pSB6A1), (b) PBAD_rbs (pSB6A1), (c) PBAD_rbs_yafO_tt_Pcon_rbs_gfp (pSB6A1), (d) Pcon_rbs_gfp (pSB6A1) were inoculated on LB agar medium (A) (ampicillin 50 microg / mL) and LB agar medium with 0.2% arabinose (B) (ampicillin 50 microg / mL), and incubated at 37°C.

2. Toxin-Antitoxin Assay

Four types of E. coli containing plasmids shown in design page were inoculated into liquid medium. When turbidity reached 0.03, arabinose was added (final concentration 0.02%) to each culture. After two hours incubating with arabinose, IPTG was also added (final concentration 0.02%). Time dependent change of RFU (relative fluorescence units) and turbidity is shown in Fig. 4 Graph (A) shows that even though E. coli containing plasmid (a) has yafN gene, it couldn’t recover the cell growth as well as one containing plasmid (c). From graph (B), no recovery of RFU could be seen on E coli containing plasmid (a), and its time dependent change was similar to that of turbidity.

Each culture contains ampicillin (50 microg/mL) and kanamycin (50 microg/mL). Arabinose and IPTG were added so that the final concentration is 0.02% and 2 mmol/L. Graph (A) shows time dependent change of turbidity, and graph (B) shows time dependent change of RFU of GFP.

Sequence and Features