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).
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 the 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 above, we determined parameters for the induction mechanism by fitting the results to the following Hill equation (Fig. 5). 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 (Hoagland et al., 1950). Our part can sense the concentration below 1 mM, too (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.
| Parameter | Value |
| α | 1700 |
| β | 5.8 |
| m | 160 |
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.
For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/phoA_Promoter_Assay our work in Tokyo_Tech 2013 wiki].
In the absence of a lab, The University of Florida 2020 iGEM team offers literature to further understand and improve part BBa_K1139201. To increase the expression of Part BBa_K1139201 designed by iGem Tokyo 2013 Team, the RiboJ insulator sequence can be added between the pPhoA promoter and the downstream sequences. Insulators protect against unexpected interactions between neighboring sequences in a genetic circuit (Clifton 2). A common insulator, RiboJ, is made up of the sTRSV-ribozyme, along with a subsequent 23-nucleotide hairpin sequence (Luo 3). The hairpin structure helps expose the ribosome binding site, so that translation can be increased for the downstream sequence transcripts (Luo 3). Therefore, insulators, and specifically, the RiboJ sequence, can be used to increase the efficacy of gene and protein expression in future experimental genetic constructs.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 754
| None |