Team:TUDelft/SDP Conclusion

=Conclusion=

The goal of the Self Destructive Plasmid subproject was to create a plasmid that could be induced to destruct itself. It would do so by expressing an endonuclease that would recognise and cut a sequence in the same plasmid. This linearised DNA was expected to be automatically degraded by the cell.

The final goal of self destruction was not reached. However, a 30 basepair I-SceI homing endonuclease restriction site has been biobricked and shown to be restricted by that endonuclease. It has been combined with an inducible GFP-LVA reporter gene, which has also been shown to work.

The problem is the I-SceI homing endonuclease. First a part from the registry was used, that turned out to be T4 DNA ligase instead of I-SceI homing endonuclease. Construction of this part was done by assembling more basic parts. But the gene for the I-SceI enzyme turned out to have an LVA degradation tag attached to it. This shortens the life time of the protein inside the cell considerably and possibly prevents normal folding. It is hypothesised that disfunctional I-SceI is the reason that the expected Self Destruction did not occur. However, a contribution to the registry was made by documenting the anomalies in the biobricks containing the I-SceI homing endonuclease.