Difference between revisions of "Part:BBa K1139201"

Revision as of 12:12, 27 September 2013

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) 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 the 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.

Compared to OUC-China’s phosphate sensor part including phoB promoter (Fig. 4), our phosphate sensor part shows clearer result (Fig. 3).

Fig. 3. Our phoA promoter assay result

Fig. 4. 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. 5), we identified m and the hill coefficient. Those parameters (Tab. 1) will be used in our future modeling. We also identified maximum GFP production rate in this construct. 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. 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)

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