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

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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"  
 
width="270px" height="90px" alt="methods"></div>
 
width="270px" height="90px" alt="methods"></div>
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<p>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>i.</p>
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<p>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>.</p>
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<img src="https://static.igem.org/mediawiki/2016/2/27/T--HokkaidoU_Japan--kill-switch_construct3.png"
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<p>Fig.1 The construct for kill-switch system</p>
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<table style="border-style: none">
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<tr align="center">
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<td style="border-style: none;">
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<td style="border-style:none; float:center"><img src="https://static.igem.org/mediawiki/2016/2/27/T--HokkaidoU_Japan--kill-switch_construct3.png" alt="construction" height="300px" width="auto"></td>
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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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<p>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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<p>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;
 
<p>1. IPTG induction</p>
 
<p>1. IPTG induction</p>
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<p>2. incubation at 37℃</p>
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<p>2. incubation at 37Ž</p>
 
<p>3. measurement of OD600 every hour</p>
 
<p>3. measurement of OD600 every hour</p>
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<p>If the system works correctly, OD600 after IPTG induction should decrease every hour, whereas the negative controls wouldn’t show apparent decrease in OD600.</p>
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<p>If the system works correctly, OD600 after IPTG induction should decrease every hour, whereas the negative controls wouldnft show apparent decrease in OD600.</p>
  
  

Revision as of 12:35, 17 October 2016

Team:HokkaidoU Japan - 2016.igem.org

 

Team:HokkaidoU Japan

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overview

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.colii would be vital damage for E.colii 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.colii containing harmful substances like heavy metal ion inside.

methods

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 E.coli.

construction

Fig. 1. The construct for kill-switch system

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;

1. IPTG induction

2. incubation at 37Ž

3. measurement of OD600 every hour

If the system works correctly, OD600 after IPTG induction should decrease every hour, whereas the negative controls wouldnft show apparent decrease in OD600.

reference