Our first piece of evidence that we are likely to get many combinations from our plasmid arrangements is the following image:

Within each quadrant, the colonies here have been transformed with exactly the same plasmid, yet generate different colours. We have not sequenced all of these colonies since transformation, however they were transformed with a sequenced plasmid. This made us realise that you could never replicate one of the bacteria we send as a key by simply sequencing and transforming the bacteria with this plasmid. This increases security of our system. In order to investigate this idea further, we wanted to check that once the bacteria have developed a colour, that level stays more or less the same. We took a colony containing P₄-sRFP, resuspended it in water then pipetted out 3ul into each spot shown below. The consistency of these colours shows that our results are reproducible enough to maintain access to whatever it is you have locked, yet not reproducible enough that anyone with the plasmid sequence could gain access. Success!

On top of this colour variation, we successfully characterised a range of gRNAs that give different repression efficiency as shown below. In the future we could extend this to more gRNAs which would give more possibilities of colour intensity.