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 improved a phosphate sensor part since the existing phosphate sensor part (OUC-China 2012, BBa_K737024) did not have sufficient data.

We constructed this improved part (Fig. 1) by amplifying the phoA promoter region of E. coli (MG1655) and ligating this phoA promoter (BBa_K1139200) upstream of the promoterless GFP generator (BBa_I751310). This phoA promoter is the inducible promoter of the alkaline phosphatase gene (phoA) derived from E. coli (Dollard et al., 2003). This promoter is repressed by high phosphate concentrations (Shinagawa et al., 1983; Hsieh et al., 2010) (Fig. 2).

Fig. 1. Our improved part: BBa_K1139201

Fig. 2. Regulation of the phoA promoter

By an induction assay, this part was confirmed to be repressed by the increase in phosphate concentration.

Compared to OUC-China’s phosphate sensor part including phoB promoter (Fig. 4), our phosphate sensor part showed a clearer result (Fig. 3) (Note that the scales of the vertical axis are different between the two results).

• Titech2013 parts K1139201 main Fig4.png

  Fig. 3. Our induction assay result for our phoA promoter

• Titech2013 parts K1139201 main Fig3.png

  Fig. 4. Our induction assay result for OUC-China 2012's phoB promoter

From our results explained above, we determined parameters for the induction mechanism. By fitting the results to the following Hill equation (Fig. 5), we identified the parameters for the induction mechanism. α denotes the maximum GFP expression rate in this construct. m denotes the phosphate concentration at which the GFP expression rate is half of α. β denotes the hill coefficient. Those parameters (Tab. 1) can be used in future modeling.

Plants are reported to be in phosphate starvation when its concentration is below 1 mM (D. Hoagland et al., 1950). Our part can sense also the concentration below 1 mM (Fig. 6). Therefore, we believe our improved part can be applied to agricultural field. 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.

Fig. 5. Equation for the induction mechanism

We set the parameters as follows:(Tab. 1)

Parameter	Value
α	720
β	3.3
m	190

Tab. 1. Determined parameters
α　denotes the maximum GFP expression rate in this construct.
m denotes the phosphate concentration at which the GFP expression rate is half of α.
β denotes the hill coefficient.

The result of our model is shown in Fig. 6.

Fig. 6. A model with fitting the results of our assay

For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/phoA_Promoter_Assay our work in Tokyo_Tech 2013 wiki].

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