Metabolic kill switch
KillerRed and KillerOrange are fluorescent proteins which, when irradiated with green and blue light respectively, generate reactive oxygen species (ROS).
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).
Method
Preliminary experiments were performed to calibrate the tecan reader. An OD of 0.4 on the cuvette reader was used as the optimum level of growth to induce protein production, this corresponded to 0.26 on the tecan reader.
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. Two flasks were inoculated with KillerOrange and two were innoculated with KillerRed and the final flask was an RFP control in the pSB1C3 plasmid. Optical density was measured every 1.5 hours until it had reached 0.26 on the tecan reader, then 100µl of 0.1M IPTG was used to induce the protein production. For each kill switch one culture was induced and one was not to act as a comparison as we know that the T7 promoter we are using is leaky.
The cultures are then incubated at 37 degrees 220rpm overnight). We also took 5ml of the culture and let it incubate at 4 degrees overnight. The cultures were then plated out and colonies allowed to grow overnight. The plates were irradiated with white light for 8 hrs checking CFU’s every hour. The liquid culture was run through the FACS machine to give a cell count and using a live dead dye.
Results
Enzymatic
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.
Lysozyme EnzChek assaying kit is used to measure lysozyme activity in solution where an increase in fluorescence is proportional to lysozyme activity.
Method
Results
DNA degradation
DNase 1 is an endonuclease that non-specifically cleaves DNA. We are creating a kill switch with DNase 1 to address the foremost problem associated with Biosafety - lateral gene transfer. The DNase 1 kill switch, on induction, will degrade DNA and kill the cell.
Method
Results
Continuous culture
To performed a continuous culture to test the longevity of a kill switch in a population we have used a ministat developed in the Dunham lab at the University of Washington (Miller et al). 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. Samples were taken each day for a week then every two days for the next week. We then tested the kill switches to determine if they were still functional.
Genome integration
While plasmids are widely used to carry genetic parts, integration into the host genome
could prove a more robust approach to introducing genes into organisms. Genome
integration removes the need for a selectable antibiotic resistance marker as the parts
will be faithfully replicated and the variability of copy number is removed. We are
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.
References
Bulina, M. E. et al., 2006. A genetically encoded photosensitizer. Nature Biotechnology.24(1).
Sarkisyan, K. S. et al, 2015. KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light. PLoS ONE.10(12)
Miller, A. W. et al, 2013. Design and Use of Multiplexed Chemostat Arrays. Journal of Visualised Experiments. (72).
