Difference between revisions of "Team:HokkaidoU Japan/Kill switch"

 
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{{Team:HokkaidoU_Japan/CSS}}
 
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<div id="Overview"><img src="https://static.igem.org/mediawiki/2016/f/fa/T--HokkaidoU_Japan--overview.png"  
 
<div id="Overview"><img src="https://static.igem.org/mediawiki/2016/f/fa/T--HokkaidoU_Japan--overview.png"  
 
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<td style="border-style: none;"><img src="https://static.igem.org/mediawiki/2016/3/3b/T--HokkaidoU_Japan--KillSwitch_image.png" alt="kill awitch" height="300px" width="250px" style="float:left"></td>
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<td style="border-style: none"; align="center"><span class="small">Fig. 1. Expected effect of the new cell-death system <br>as an application of SAP</span></td>
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<span class="nomal2">
 
<span class="nomal2">
  
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We came up with an idea of utilizing SAP for cell death system. To be specific, we expect SAP which is excessively accumulated inside of <span style="font-style: italic">E.coli</span>i would be vital damage for <span style="font-style: italic">E.coli</span>i by disturbing its metabolism or catabolism, for example. In addition, this system is expected to cause cell death without cell lysis, which is a desirable feature when you want to handle <span style="font-style: italic">E.coli</span>i containing harmful substances like heavy metal ion inside.
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We came up with an idea of utilizing SAP for cell death system. To be specific, we expect SAP which is excessively accumulated inside of <span style="font-style: italic">E. coli</span> to be a vital damage for <span style="font-style: italic">E. coli</span> by disturbing its metabolism or catabolism, for example. In addition, this system is expected to cause cell death without cell lysis, which is a desirable feature when you want to handle <span style="font-style: italic">E. coli</span> containing harmful substances like heavy metal ion inside.
  
 
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<div id="Methods"><img src="https://static.igem.org/mediawiki/2016/2/2c/T--HokkaidoU_Japan--methods.png"  
 
<div id="Methods"><img src="https://static.igem.org/mediawiki/2016/2/2c/T--HokkaidoU_Japan--methods.png"  
 
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width="270px" height="90px" alt="methods"></div>
 
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For our new cell death system, we designed a construct shown in fig.1. The construct has a lactose promoter upstream of RBS and SAP coding region, thus we can control the timing of switching-on the device. We ordered the parts of DNA from IDT, and after subcloning, we put the piece of DNA on pSB1C3 vector and transformed it into <span style="font-style: italic">E.coli</span>.
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For our new cell death system, we designed a construct shown in fig.2. The construct has a lactose-inducible promoter upstream of RBS and SAP coding region, thus we can control the timing of switching on the device. We ordered the parts of DNA from IDT, and after subcloning, we put the piece of DNA on pSB1C3 vector and transformed <span style="font-style: italic">E. coli</span> with it.
  
 
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<td style="border-style: none"; align="center"><h2>Fig. 1. The construct for kill-switch system</h2></td>
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<td style="border-style: none"; align="center"><span class="small">Fig. 2. The construct for kill switch system</span></td>
 
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Unfortunately we didnft have enough time to assess this cell death system, but we planned to assess the effect of this biodevice according to the following protocol;
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Unfortunately we didn't have enough time to assess this cell death system, but we planned to assess the effect of this biodevice according to the following protocol;
 
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<li>IPTG induction</li>
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<li>IPTG induction.</li>
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<li>Incubation at 37&deg;C</li>
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<li>Incubation at 37&deg;C.</li>
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<li>Measurement of OD<sub>600</sub> every hour</li>
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<li>Measurement of OD<span class="sitatuki">600</span> every hour.</li>
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<li>If the system works correctly, OD<sub>600</sub> after IPTG induction should decrease every hour, whereas the negative controls wouldnft show apparent decrease in OD<sub>600</sub>.</li>
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<li>If the system works correctly, OD<span class="sitatuki">600</span> after IPTG induction should decrease every hour, whereas the negative controls wouldn't show apparent decrease in OD<span class="sitatuki">600</span>.</li>
 
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<div id="Reference"><img src="https://static.igem.org/mediawiki/2016/8/86/T--HokkaidoU_Japan--reference.png"  
 
<div id="Reference"><img src="https://static.igem.org/mediawiki/2016/8/86/T--HokkaidoU_Japan--reference.png"  
 
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[1] Riley JM, Aggeli A, Koopmans RJ, McPherson MJ, (2008) Bioproduction and Characterization of a pH Responsive Self-Assembling Peptide. Biotechnol Bioeng. 103(2): 241-51<br>
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[2] Kyle　S, Aggeli A,Ingham E, McPhersona MJ, (2010) Recombinant self-assembling peptides as biomaterials for tissue engineering．Biomaterials. 31(36): 9395-9405<br>
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[3] Prakash A,Parsons SJ, Kyle S, McPherson MJ (2012) Recombinant production of self-assembling β-structured peptides using SUMO as a fusion partner. Microbial Cell Factories. 2012, 11:92. <br>
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Latest revision as of 19:48, 19 October 2016

Team:HokkaidoU Japan - 2016.igem.org

 

Team:HokkaidoU Japan

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overview
kill awitch
Fig. 1. Expected effect of the new cell-death system
as an application of SAP
We came up with an idea of utilizing SAP for cell death system. To be specific, we expect SAP which is excessively accumulated inside of E. coli to be a vital damage for E. coli by disturbing its metabolism or catabolism, for example. In addition, this system is expected to cause cell death without cell lysis, which is a desirable feature when you want to handle E. coli containing harmful substances like heavy metal ion inside.
methods
For our new cell death system, we designed a construct shown in fig.2. The construct has a lactose-inducible promoter upstream of RBS and SAP coding region, thus we can control the timing of switching on the device. We ordered the parts of DNA from IDT, and after subcloning, we put the piece of DNA on pSB1C3 vector and transformed E. coli with it.
construction
Fig. 2. The construct for kill switch system

Unfortunately we didn't have enough time to assess this cell death system, but we planned to assess the effect of this biodevice according to the following protocol;
  1. IPTG induction.
  2. Incubation at 37°C.
  3. Measurement of OD600 every hour.
  4. If the system works correctly, OD600 after IPTG induction should decrease every hour, whereas the negative controls wouldn't show apparent decrease in OD600.

reference
[1] Riley JM, Aggeli A, Koopmans RJ, McPherson MJ, (2008) Bioproduction and Characterization of a pH Responsive Self-Assembling Peptide. Biotechnol Bioeng. 103(2): 241-51
[2] Kyle　S, Aggeli A,Ingham E, McPhersona MJ, (2010) Recombinant self-assembling peptides as biomaterials for tissue engineering．Biomaterials. 31(36): 9395-9405
[3] Prakash A,Parsons SJ, Kyle S, McPherson MJ (2012) Recombinant production of self-assembling β-structured peptides using SUMO as a fusion partner. Microbial Cell Factories. 2012, 11:92.