pYTK001 was mixed with a custom forward/reverse primer pair and PCR reaction carried out according to protocol using Phusion polymerase. The produced solution was purified using gel purification followed by gel purification kit from Thermo Fisher, and the resultant linear fragment was cyclised using Gibson assembly protocols, followed by transformation into competent E.coli strain DH5-alpha. The plated cells were inoculated, grown overnight and plasmids extracted using Thermo Fisher plasmid miniprep kit and protocol. From the extracted plasmids a sample was sent to sequencing, while the remainder was used to transform again, and following overnight growth, inoculated again, followed by equalizing the OD of each sample. Alongside a positive control consisting of unmodified pYTK001 sfGFP-containing plasmid, a negative control of transformed pYTK002 (without GFP) in DH5-alpha E.coli, the fluorescence was measured with three replicates of each RBS using a plate reader and the strength of each RBS was measured.