This detection method requires two biosensors, one control biosensor to which only water will be applied and another for the actual sample. Our DNP biosensor uses crude cell lysate from cells expressing the sequence confirmed laccase CBD fusions which is then washed with PBS to purify the enzyme. A solution containing 0.2mM ABTS and 100mM sodium acetate buffer at pH5 is then added to the biosensor and is freeze-dried onto paper. A dark blue colour in the control will appear as soon as the biosensor is rehydrated. A difference in the blue colour between the two biosensors will indicate the presence of DNP. In the presence of DNP, the colour change will be slower and produce a much lighter blue, which when read with a spectrophotometer at 420 nm has a lower absorbance than the control. This is also a binary detection which should be visible with the naked eye.

The laccase we are using is a copper-dependent tvel5 laccase from Trametes versicolor encoded by the gene lccl. To make our fusions compatible, we used biobrick assembly standard RFC25 ². The RFC25 prefix and suffix were added to the gene and all illegal restriction sites (EcoRI, SpeI, AgeI, NotI, NgoMIV and XbaI) were removed. The sequence was codon optimised and ordered as a gBlock. It was then digested and ligated into the pSB1C3 backbone and transformed into an Escherichia coli chassis.