Difference between revisions of "Part:BBa K1139201"

Revision as of 06:02, 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) 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.

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 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. 6). Therefore, our improved part is useful for our farming circuit. We also identified maximum GFP production rate in this construct.

Fig. 5. Equation for our mathematical model

We set the parameters as follows:

Parameter	Value
α	720
β	3.3
m	190

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

Fig. 6. Result of our mathematical model

For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/RM-lac_Hybrid_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

@@ Line 12: / Line 12: @@
 By an induction assay, this part was confirmed to be repressed by the increase in phosphate concentration. <br>
-Compared to OUC-China’s phosphate sensor part including <i>phoB</i> promoter (Fig. 3), our phosphate sensor part shows clearer result (Fig. 4).
+Compared to OUC-China’s phosphate sensor part including <i>phoB</i> promoter (Fig. 4), our phosphate sensor part shows clearer result (Fig. 3).
-[[Image:titech2013_parts_K1139201_main_Fig3.jpg|thumb|none|300px|<b>Fig. 3.</b> OUC-China 2012’s <i>phoB</i> promoter assay result (converted to bar chart)]]
+[[Image:titech2013_parts_K1139201_main_Fig4.jpg|thumb|left|300px|<b>Fig. 3.</b> Our <i>phoA</i> promoter assay result]]
-[[Image:titech2013_parts_K1139201_main_Fig4.jpg|thumb|none|300px|<b>Fig. 4.</b> Our <i>phoA</i> promoter assay result]]
+[[Image:titech2013_parts_K1139201_main_Fig3.jpg|thumb|none|300px|<b>Fig. 4.</b> OUC-China 2012’s <i>phoB</i> 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 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. 6).  Therefore, our improved part is useful for our farming circuit.  We also identified maximum GFP production rate in this construct.<br>