Difference between revisions of "Part:BBa K1949032"

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<partinfo>BBa_K1949032 short</partinfo>
 
<partinfo>BBa_K1949032 short</partinfo>
  
<span style="margin-left: 10px;">This part consists of P<i>lac</i>(<partinfo>BBa_R0010</partinfo>) + RBS(<partinfo>BBa_B0034</partinfo>) + <i>yafO</i>(<partinfo>BBa_K1949030</partinfo>).<br>
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[[Image:Yafoyafn2.png|thumb|center|400px| Antitoxin YafN inhibit function of toxin YafO]]<br>
  
<span style="margin-left: 10px;">Toxin-antitoxin (TA) systems are cassettes consisting of stable protein, toxin which function as mRNA interferase and unstable protein, antitoxin which destroys function of toxin. Many TA systems exist in the <i>E. coli</i> genome. They can inhibit cell growth or induce cell killing. YafO is one of the toxin and is co-expressed with YafN.<br>
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<span style="margin-left: 10px;">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 <i>E. coli</i> 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.<br>
  
 
<span style="margin-left: 10px;">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.<br>
 
<span style="margin-left: 10px;">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.<br>
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<span style="margin-left: 10px;">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.<br>
 
<span style="margin-left: 10px;">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.<br>
  
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[[Image:Yafno4.png|thumb|center|400px| Fig.1 Construction of the Experiment ]]<br>
  
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[[Image:yafon5.png|thumb|center|400px| Fig.2 Construction of the Experiment ]]<br>
  
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===Characterization===
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<span style="margin-left: 10px;">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.
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====1. Confirming YafO Function as Toxin on Agar Medium====
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<span style="margin-left: 10px;">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.
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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.
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[[Image:yafon1.png|thumb|center|400px| Fig.3 Confirming YafO function as toxin on agar medium ]]<br>
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Each E. coli containing  (a) PBAD_<i>rbs_yafO</i> (pSB6A1), (b) PBAD_<i>rbs</i> (pSB6A1), (c) PBAD_<i>rbs_yafO_tt</i>_Pcon_<i>rbs_gfp</i> (pSB6A1), (d) Pcon_<i>rbs_gfp</i> (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.
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====2. Toxin-Antitoxin Assay====
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<span style="margin-left: 10px;">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.
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[[Image:yafon2.png|thumb|center|400px| Fig.4-(a) toxin-antitoxin assay ]]<br>
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[[Image:yafon3.png|thumb|center|400px| Fig.4-(b) toxin-antitoxin assay ]]<br>
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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.
  
  

Revision as of 09:00, 17 October 2016


PBAD-rbs-yafO

Antitoxin YafN inhibit function of toxin 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.

Fig.1 Construction of the Experiment

Fig.2 Construction of the Experiment

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal XhoI site found at 1571
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961