Difference between revisions of "Part:BBa K1139201"

Revision as of 16:41, 27 October 2013

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).

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

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

@@ Line 4: / Line 4: @@
 PphoA is a promoter that is activated by PhoB-phosphorylated when phosphate concentration is low. <i>GFP</i> is a reporter.
-We constructed this part by ligating <i>phoA</i> promoter (<partinfo>BBa_K1139200</partinfo>) upstream of promoterless GFP generator (<partinfo>BBa_I751310</partinfo>). <br>
+We <b>improved</b> a phosphate sensor part since the existing phosphate sensor part (OUC-China 2012, <partinfo>BBa_K737024</partinfo>) did not have sufficient data.
-We <b>improved</b> a phosphate sensor part since the existing phosphate sensor part (OUC-China 2012, <partinfo>BBa_K737024</partinfo>) did not have sufficient data. We constructed this part by amplifying the <i>phoA</i> promoter region of <i>E. coli</i> (MG1655) and ligating it upstream of the <i>GFP</i> part (Fig. 1). This <i>phoA</i> promoter is the inducible promoter of the alkaline phosphatase gene (<i>phoA</i>) from <i>E. coli</i> (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).<br>
+We constructed this improved part (Fig. 1) by amplifying the <i>phoA</i> promoter region of <i>E. coli</i> (MG1655) and ligating this <i>phoA</i> promoter (<partinfo>BBa_K1139200</partinfo>) upstream of the promoterless GFP generator (<partinfo>BBa_I751310</partinfo>). This <i>phoA</i> promoter is the inducible promoter of the alkaline phosphatase gene (<i>phoA</i>) derived from <i>E. coli</i> (Dollard et al., 2003). This promoter is repressed by high phosphate concentrations (Shinagawa et al., 1983; Hsieh et al., 2010) (Fig. 2).
 [[Image:titech2013_parts_K1139201_main_Fig1.jpg|thumb|center|300px|<b>Fig. 1.</b> Our improved part: <partinfo>BBa_K1139201</partinfo>]]
-[[Image:titech2013_parts_K1139201_main_Fig2.jpg|thumb|center|300px|<b>Fig. 2.</b> Mechanism of <i>phoA</i> promoter]]
+[[Image:titech2013_parts_K1139201_main_Fig2.jpg|thumb|center|300px|<b>Fig. 2.</b> Regulation of the <i>phoA</i> promoter]]
 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. 4), our phosphate sensor part shows clearer result (Fig. 3).
+Compared to OUC-China’s phosphate sensor part including <i>phoB</i> 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).
-[[Image:titech2013_parts_K1139201_main_Fig4.jpg|thumb|left|360px|<b>Fig. 3.</b> Our <i>phoA</i> promoter assay result]]
+[[Image:titech2013_parts_K1139201_main_Fig4.jpg|thumb|left|360px|<b>Fig. 3.</b> Our induction assay result for our <i>phoA</i> promoter]]
-[[Image:titech2013_parts_K1139201_main_Fig3.jpg|thumb|none|380px|<b>Fig. 4.</b> OUC-China 2012’s <i>phoB</i> promoter assay result (converted to bar chart)]]
+[[Image:titech2013_parts_K1139201_main_Fig3.jpg|thumb|none|380px|<b>Fig. 4.</b> Our induction assay result for OUC-China 2012's <i>phoB</i> promoter]]
 <br>
-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 <i>E. coli</i> that could increase plant growth by synthesizing several plant hormones depending on the soil environment. <br>
+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 <i>E. coli</i> that could increase plant growth by synthesizing several plant hormones depending on the soil environment. <br>
 [[Image:titech2013_parts_K1139201_main_Fig5.jpg|thumb|center|300px|<b>Fig. 5.</b> Equation for the induction mechanism]]
@@ Line 33: / Line 38: @@
 |&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;m||190
 |}
+Tab. 1. Determined parameters <br>
+<font size ="1"><b>α　denotes the maximum GFP expression rate in this construct. <br>m denotes the phosphate concentration at which the GFP expression rate is half of α. <br>β denotes the hill coefficient.</b></font size>
 The result of our model is shown in Fig. 6.<br>