Part:BBa_K2447000

Extracellular phosphate sensor with GFP reporter

PhoB and PhoR proteins are part of the Pho regulon in E. coli. When a low concentration of extracellular phosphate ions is present, PhoR can phosphorylate PhoB to form an active phosphorylated-PhoB. PhoB promoter is activated as a result of active binding of phosphorylated-PhoB, resulting in downstream expression of GFP. When a high concentration of extracellular phosphate ions is present, PhoR will dephosphorylate PhoB, and therefore inactivating it, and repressing the PhoB promoter for GFP expression.

Figure 1: PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP.

Improvement over previous iGEM part BBa_K116404 (NYMU Taipei 2008)

The two parts (Bba_K2447000 & BBa_K116404) listed below are inserted into pBbE2k backbone and subsequently characterized in E. coli MG 1655. These cells were grown in LB medium before resuspension in MOPS medium (a minimal nutrient medium) for characterization with a micro-plate reader. Varying concentrations of phosphate ions from 0 to 1000 uM were added and GFP expression was monitored.

By replacing the weaker RBS BBa_B0032 of the original part with a stronger RBS BBa_B0034, we have successfully constructed an improved phosphate sensor-GFP reporter. Our part shows, on average, a 40-fold increase in GFP expression (Figure 3) when compared to the previous version of the construct (http://2008.igem.org/Team:NYMU-Taipei/Project/Phosphate). The original phosphate construct is also insensitive to high phosphate concentrations above 50 µM where similar levels of GFP expression are observed (Figure 4). Unlike the previous construct, our improved phosphate construct is much more sensitive to various phosphate concentrations from 0 to 1000 µM, particularly at phosphate concentrations above 50 µM.

Figure 2: Side by side comparison of our improved construct Bba_K2447000 & the original construct BBa_K116404. Our proposed part exhibited greater GFP expression at every phosphate concentration and exhibited much higher sensitivity to phosphate levels above 50 uM.

Figure 3: Our improved phosphate construct is much more sensitive to phosphate concentrations above 50 uM, unlike the original construct.

Figure 4: GFP expression by the original phosphate construct designed by the Taiwanese team.

Experience

Utilizing the modified phosphate range and characterization protocol (See: Modified Protocol under Pho), we characterized the BBa_K2447000 phosphate sensor. The characterization curve (Figure 1) showed a linear, negative trend throughout phosphate concentrations ranging from 0-100μM. This data closely paralleled the predictive ODE model created by our 2020 team and the previous characterization data by NUS Singapore iGEM 2017.

Figure 5: Characterization curve for BBa_K2447000 for phosphate concentrations between 0μM and 100μM.

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 1162