Part:BBa_K1139201

PphoA-GFP-TT

PphoA is a promoter that is activated by PhoB-phosphorylated when phosphate concentration is low. GFP is a reporter.

We constructed this part by ligating phoA 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 µM.

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)

From our results explained above, we determined parameters for the induction mechanism. By fitting the results to the following Hill equation (Fig. 6), we identified K and the hill coefficient. Those parameters (Tab. 1) will be used in our future modeling. 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 (Fig. 7). Therefore, our improved part is useful for our farming circuit. We also identified maximum GFP production rate in this construct.

Fig. 6. Equation for our mathematical model

We set the parameters as follows:

File:Titech2013 parts K1139201 Tab1false.jpg

Tab. 1. Parameters and Values

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

Fig. 7. Result of our mathematical model

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