Team:HokkaidoU Japan/Kill switch

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.

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