Difference between revisions of "Part:BBa K1949032"

Latest revision as of 18:06, 19 October 2016

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 ribosome-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

In this experiment, E. coli XL 1-Blue strains were introduced the following plasmids:
(a) PBAD ‐ rbs ‐ yafO (pSB6A1)
(b) PBAD ‐ rbs (pSB6A1)
(c) PBAD ‐ rbs ‐ yafO ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1)
(d) Pcon ‐ rbs ‐ gfp (pSB6A1)
These E. coli were inoculated on agar plates with or without 0.2% arabinose, and incubated at 37°C. The result is shown in Fig. 1. In this figure, (A) shows agar plate without arabinose and (B) shows agar plate with 0.2% arabinose. E. coli containing plasmid (a) and one containing plasmid (c) couldn’t form any colonies on agar plate (B), although all types of E. coli was able to form colonies on agar plate (A). From this result, cell growth was inhibited by inducing expression of YafO. Particularly, on agar plate containing arabinose (A) E. coli containing plasmid (c) formed fluorescent colonies like E. coli containing plasmid (d), and on agar plate containing no arabinose (B) the E. coli didn' t form any colonies like E. coli containing plasmid (a) . This result insists that genes on plasmid (c) were working for sure.

Fig.1 Confirming YafO function as toxin on agar plates

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 plate (A) (ampicillin 50 microg / mL) and LB agar plate with 0.2% arabinose (B) (ampicillin 50 microg / mL), and incubated at 37°C.

2. Toxin-Antitoxin Assay

In this experiment, E. coli XL 1-Blue strains were transformed by the following plasmids:
(a) PBAD ‐ rbs ‐ yafO ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1), Plac <>PBAD ‐ rbs ‐ yafO (pSB6A1)
(b) PBAD ‐ rbs (pSB6A1)
(c) PBAD ‐ rbs ‐ yafO ‐ tt ‐ Pcon ‐ rbs ‐ gfp (pSB6A1)
(d) Pcon ‐ rbs ‐ gfp (pSB6A1)
These E. coli were inoculated in liquid media, respectively. 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 plasmids (a) has yafN gene, it didn’t recover the cell growth as well as one containing plasmids (c). From graph (B), no recovery of RFU could be seen on E coli containing plasmids (a), and its time-dependent change was similar to that of turbidity.

Fig.2-(a) toxin-antitoxin assay

Fig.2-(b) toxin-antitoxin assay

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

Sequence and Features