E. coli that glows in paucity of phosphate - at a glance

A. E. coli engineered with BBa_K1682013 or BBa_K1682015 functions as a phosphate biosensor. High concentrations of phosphate indirectly represses PphoA and PphoBR and decreases the expression of GFP.		B. The phosphate sensing promoters BBa_K1682013 and BBa_K1682015 can both detect a gradient of phosphate concentrations. Its activities at 0 and 300 μM were reported in Relative Fluorescence Level (a.u.).

• Phosphate associate with essential growth factors for plant root development as well as seed production.
• We improved on P_phoA, P_phoBR. The data provided dynamic range of these promoters.

P_phoA , P_phoBR and our engineered phosphate sensor BBa_K1682013 and BBa_K1682015 - the full story

Phosphorus is an essential plant macronutrient as it associates with various growth factors for plant root development as well as seed production. Deficiency in phosphorus will result in stunted plant growth. Our aim is to engineer a phosphate sensor in Escherichia coli and detect phosphate level in soil. To this end, we obtained different sequences for P_phoA or P_phoBR and fused it with gfp (gfpmut3b). The promoters are activated under low phosphate condition, so green fluorescence will be produced to show phosphate level in soil.

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P_phoA (BBa_K1139201) is first characterized and BioBricked by Tokyo Tech 2013 iGEM team. P_phoBR (BBa_K737024) is first characterized and BioBricked by OUC-China 2012 iGEM team. To provide more characterization data on these two promoters, we improved these promoters by obtaining different sequences.

Endogenous phosphate sensing system in E. coli

Figure 1. The Pho phophorous sensing system in E. coli.

E. coli has multiple native phosphorus sensing and regulation systems that we could use in the construct. Among them, we chose the PhoR/PhoB two-component system (TCS). It contains a sensory histidine kinase PhoR and a partner DNA-binding response regulator PhoB. PhoR is activated under low phosphate concentration, which will then phosphorylate PhoB. The phospho-PhoB is then capable of activating expression of the Pho regulon genes, one of the examples is phoA. In high phosphate concentration, phoR is turned into an inhibitory state, which interferes with phosphorylation of PhoB. PhoB is, thus, not capable of activating expression of phoA.

*The above text is our summarized understanding on phosphate-sensing system using information from Hsieh & Wanner (2010). Please refer to our references section below for the reference cited.

Design and Testing of phosphate sensing Device

Figure 2. Construction and Testing of P_phoA and P_phoBR. A) Constructs of BBa_K1682013 and BBa_K1682015. P_phoA and P_phoBR ligated with gfp (gfpmut3b) B) Characterization of P_phoA and P_phoBR in M9 minimal medium. Promoter's activities were reported in Fluorescence per Biomass. Error bar present SEM from 3 biological replicates.

To construct a phosphate-sensing device, we cloned the promoters P_phoA and P_phoBR, and fused it with a GFP reporter BBa_I13504 in BioBrick RFC10 standard. The activity of promoter can then be reported by the fluorescence level under different phosphate concentrations.

The activities of P_phoA (BBa_K1682013) and P_phoBR (BBa_K1682015) in different phosphate concentrations were measured in Fluorescence per Biomass, following a modified protocol (see below) from Tokyo Tech 2013 iGEM team. They were found to be falling in a dynamic range of 0 - 300 μM of phosphate. In their dynamic range, there is a 2.99 fold change in Fluorescence for P_phoA and a 1.88 fold change in Fluorescence for P_phoBR.

Comparing activities of P_phoA and P_phoBR

Figure 3. Comparison between P_phoA and P_phoBR GFP expression.

We selected P_phoA for our construct. We compared the activity difference of two promoters for 0 and 300 μM of phosphate, as they are the minimum and maximum dynamic range suggested by Tokyo Tech 2013 iGEM team. Having The fold change of P_phoA GFP is 2.99, which is greater than 1.88 of P_phoBR. Therefore, we chose P_phoA as it has a greater range, so it is supposed to have larger difference within a certain range.

Materials and Methods

Please refer to our protocol page for the materials and methods used in characterization.

References

Hsieh, Y. J., & Wanner, B. L. (2010). Global regulation by the seven-component P i signaling system. Current opinion in microbiology, 13(2), 198-203.