In order to tune our bacteria so that it would behave correctly in vivo, we had to define precise, quantitative criteria that had to be met. This includes on the one hand the detection thresholds in term of bacterial cells density and lactate concentration for our AND gate, and on the other hand the azurin output level that could be reasonably obtained in the tumor from the lysis of our bacteria. As this later criterium is tightly related to the bacterial cells density that can be achieved in the tumor (the more bacterial, the more azurin is released), let us begin with focusing on bacterial cells density.

Estimating a precise distribution of cells into the tumor is not straightforward, and we had to apply assumptions that appeared reasonable to us to be able to estimate a plausible quantitative repartition of bacteria in solid tumors.