1. mcherry reporter is changed into EGFP as the consideration of the strength of EGFP is stronger than mcherry, meanwhile EGFP is more sensitive and detectable than mcherry by microplate reader in realistic conditions. Figure 1 shows intensity of mcherry in previous BBa_K2448025(link), and we know that it is rather hard to see strength changes in the first few concentration gradients and the spike around 1mM psicose is really hard to illustrate.

Figure 1.Changes of fluorescence intensity per OD with different concentration of psicose (Data cited from BBa_K2448025)

Compared with mcherry, EGFP fluorescence intensity is 100 times stronger than mcherry. And we could distinguish differences of fluorescence per ABS600 within 5mM psicose easily, as shown in Figure 2. As a results, the detection range of our sensing circuit is wider than previous parts and our part is more sensitive.

Figure 2 Dose effect of D-psicose' s effect on pPsi promoter (Our improved data)

2. We have made some improvements in the experiment design. In addition to testify the effect of fructose on pPsi promoter in in previous BBa_K2448025 (Figure 3), we measured effect of fructose and IPTG, which make our design more complete and persuasive (Figure 4). Figure 4 shows that at the same timepoint, the fluorescence intensity varies with different sugar, and psicose is always higher than other sugar, while IPTG has a little effect to induce pPsi promoter. So we can draw the conclusion that pPsi promoter is specific to D-psicose and is a little sensitive to IPTG in this system.

Figure 3 Influence of fructose in previous part (Data cited from BBa_K2448025)

Figure 4 Effect of different sugars on pPsi promoter (Our improved data)

3. We changed the direction of reporter gene, as shown in Fig 5. In previous BBa_K2448025, The operons of PsiR (promoted by a constative promoter) and EGFP (promoted by pPsi) are in the same direction, but in our circuit they are in the reverse direction, in order to minimize read-through of terminators and to prevent homologous recombination of the plasmid itself. As this circuit will be present in chassis cells under high growth pressure, the cell or plasmid could undergo unexpected mutations to maximize its survival. This design is meant to prevent such things from happening.

Fig 5 Design of our sensing circuit (BBa_K2791004)