<pstyle="margin-top: 20px;">It is to the consensus that the -35 and -10 boxes, the binding sites of the sigma70 factor during transcription, play a major role determining the strength of the respective promoter. Previous iGEM researchers employed saturation mutagenesis on the two regions and generated the J23119 family promoters. Among these promoters with different strengths, they concluded that J23119 from the family was the strongest1. Building upon this discovery, we leave the -35 and –10 consensus sequences identical to J23119 and utilize saturation mutagenesis via GoldenGate Assembly to generate randomized sequences on the sequential context surrounding the sigma factor recognition site, as shown below. This new construct is named pP6, with its design allowing us to explore strong constitutive promoters with a larger diversity in their gene sequence. Meanwhile, our hope was that altering sequential context would yield constitutive promoters even stronger than J23119.</p>
<pstyle="margin-top: 20px;">It is to the consensus that the -35 and -10 boxes, the binding sites of the sigma70 factor during transcription, play a major role determining the strength of the respective promoter. Previous iGEM researchers employed saturation mutagenesis on the two regions and generated the J23119 family promoters. Among these promoters with different strengths, they concluded that J23119 from the family was the strongest [1]. Building upon this discovery, we leave the -35 and –10 consensus sequences identical to J23119 and utilize saturation mutagenesis via GoldenGate Assembly to generate randomized sequences on the sequential context surrounding the sigma factor recognition site, as shown below. This new construct is named pP6, with its design allowing us to explore strong constitutive promoters with a larger diversity in their gene sequence. Meanwhile, our hope was that altering sequential context would yield constitutive promoters even stronger than J23119.</p>
<pstyle="margin-top: 20px;">For successful constructs proven by sequencing, we retransform and measure GFP signal, excited using 483 nm, at 525 nm wavelength with Tecan reader for later significance tests.</p>
