An operon containing four genes encoding the naringenin biosynthetic pathway (Figure 1) was previously assembled by TU Darmstadt 2014 iGEM team (BBa_K1497016). We needed to assemble a naringenin operon to allow the genes that encode for the enzymes that produce naringenin to be implemented into DH5 alpha cells. Modifications were made to TU Darmstadt’s operon, as the second gene in the operon (TAL) needed to be manually codon optimised to be synthesised by IDT. Further the last two genes CHS and CHI in the operon were switched in the Benchling design for our operon. These gblocks were ordered from IDT with the relevant primers for amplification and detection of the gblocks. We decided to use Gibson Assembly, because it was how TU Darmstadt assembled their operon successfully and the assembly is a scarless method based on the overlaps of fragments.

The first part of this to accomplish was amplifying the pSB1C3 backbone that would take up the naringenin operon. Competent cells were then to be produced and tested using a positive control for Gibson Assembly. The gblocks synthesised by IDT were utilised in Gibson Assemblies and subsequent transformation of the naringenin operon into DH5 alpha competent cells was to be attempted. This would be tested through a series of minipreps and sequencing of transformed colonies. Alongside this a new design of the naringenin operon based on the pathway modelling, was constructed to show how different promoters with varying strengths could increase naringenin production.