Part:BBa_K1139201

PphoA-GFP-TT

We constructed this part by ligating PphoA promoter (BBa_K1139200) to the upstream of promoterless GFP generator (BBa_I751310).
We improved a phosphate sensor part since the existing phosphate sensor part (OUC-China 2012, BBa_K737024) did not have sufficient data. We constructed this part by amplifying the phoA promoter region of E. coli (MG1655) and ligating it upstream of GFP part (Fig. 1). This phoA promoter is the inducible promoter of the alkaline phosphatase gene (phoA) from E. coli (M. Dollard et al., 2003). This promoter is repressed by high concentration phosphate (H. Shinagawa et al., 1983, Y. Hsieh et al., 2010) (Fig. 2).

Fig. 1. Our improved part: BBa_ K1139201

Fig. 2. Mechanism of phoA promoter

By an induction assay, this part was confirmed to be repressed by the increase in phosphate concentration. Fig. 3 shows the result that the phoA promoter is drastically repressed at phosphate concentrations of 100 to 300 microM.

Fig. 3. Result of phoA promoter induction assay

Compared to OUC-China’s phosphate sensor part including phoB promoter (Fig. 5), our phosphate sensor part shows clearer result (Fig. 4).

Fig. 4. Our phoA promoter assay result

Fig. 5. OUC-China 2012’s phoB promoter assay result (converted to bar chart)

Moreover, plants are reported to be in phosphate starvation under the concentration of 1 mM (D. Hoagland et al., 1950). Our part can also sense the concentration below 1 mM. By combining this part with other parts, various ideas can be achieved. For instance, we have a future plan to create E. coli that could increase plant growth by synthesizing several plant hormones depending on the soil environment.
From our induction assay explained above, we set the parameters and built the mathematical model to predict how our part responds to phosphate concentration. Our model is described by the following differential equation based on Hill equation (Fig. 6).

Fig. 6. Equation for our mathematical model

We set the parameters as follows:

Tab. 1. Parameters and Values

The result of our model is shown in Fig. 7.

Fig. 7. Result of our mathematical model

This result agrees with our wet assay result. Therefore, we confirm that we constructed a credible part. Moreover, plants are reported to be in phosphate starvation under the concentration of 1 mM (D. Hoagland et al., 1950). Our part can also sense the concentration below 1 mM. Therefore, this part will lead us to our goal to create E. coli which could increase plant growth by synthesizing several plant hormones depending on the soil environment.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 754