Difference between revisions of "Part:BBa K1949022"

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Plac-rbs-yafN

YafN is an antitoxin corresponding to toxin YafO, and YafN reverses inhibition of cell growth. yafN and yafO exist as an operon, and YafN also functions as a repressor in the operon. Therefore, yafNO operon’s translation is autoregulated by antitoxin YafN. When Lon protease senses environmental stresses such as DNA damage, the operon becomes active because of degradation of YafN by Lon protease.

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 Medium

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.

Fig.4-(a) toxin-antitoxin assay

Fig.4-(b) toxin-antitoxin assay

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