KillerRed has been shown to effectively kill cells when exposed to green light (540–580 nm) and is much less effective under blue light(460–490 nm) (Bulina, 2006). KillerOrange is a mutant of KillerRed that is excited from 420-530nm that has been shown to work alongside KillerRed (Sarkisyan,2015). Our project has improved the characterisation of a KillerRed codon optimised for E. coli. We are also characterising KillerOrange in the same way. We aim to include both KillerRed and KillerOrange in the same system so as to be a more phototoxic. High levels of ROS lyse the cell but can also damage the DNA, this is an attractive prospect when developing a kill switch to reduce the risk of horizontal gene transfer (HGT).</p>

5ml overnights of the transformed E. coli with the KillerOrange and KillerRed kill switches were used to inoculate five 250ml erlenmeyer flasks covered in tin foil containing 50ml of LB 35µg/ml Chloramphenicol. The five flasks were inoculated with the following conditions. </p>

<p id="pp">Lysozyme is a common enzyme used in laboratories and the gallus gallus form is basis of our enzymatic kill switch. It is bacteriolytic when transported into the periplasm of gram negative bacteria, hydrolysing the glycosidic bond connecting N-acetylmuramic acid and N-acetylglucosamine. Under the control of a T7 promoter we can induce lysis of the cell by adding IPTG. Simply lysing the cell does not prevent HGT it may even exacerbate the problem. We have tested the liklihood of HGT by lysing a culture of cells producing RFP, incubating the lysate in a 90 degree water bath to inactivate the lysozyme. Then growing competent E. coli with the lysate will show if the competent cells take up the RFP gene.

<p id="pp">We performed a continuous culture in a ministat developed in the Dunham lab at the University of Washington (Miller et al). This was to test the longevity of our kill switches. Each ministat chamber is fed from its own media container via a peristaltic pump. The culture volume is set by the height of the effluent needle in the chamber. Our preliminary experiment used E. coli expressing RFP, we tested it under different media conditions using LB with and without salt. The flow rate was set to around 4ml/hr.</p>

investigating whether integration into the E. coli genome will affect the efficiency of our

kill switches and whether they will remain functional for longer in a continuous culture. We have used the lambda red recombination method to integrate our parts into the arsB locus. Integrating in this locus does not affect E. coli growth (reference kiko paper). We used the pKD4 plasmid as a vector to carry our parts, this had a pst1 site and two xba1 sites. Using site Q5 site directed mutagenesis kit we removed these illegal restriction sites.</p>