Part:BBa_K2447000

Extracellular phosphate sensor with GFP reporter

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

PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP

Improvement over previous iGEM part BBa_K116404 (NYMUTaipei 2008)

The two parts (Bba_K2447000 & BBa_K116404) listed below are inserted into pBbE2k backbones and subsequently characterised in E.coli MG1656. These cells are grown in MOPS medium (a minimal nutrient medium) and varying concentrations of phosphate ions from 0 to 1000 uM are added.

By replacing the weaker RBS 32 (as utilised by the Taiwanese team) with a stronger binding affinity RBS 34 as proposed by IGEM NUS team 2017, we have elucidated much stronger GFP expression and also improved the sensivity of the part to varying concentrations of phosphate ions. Previously, the Taiwanese part [http://2008.igem.org/Team:NYMU-Taipei/Project/Phosphate had reported]similar level of repressed GFP productions for phosphate concentrations above 40uM. On the other hand, our proposed phosphate sensor is sensitive to phosphate concentration above 40 uM, elucidating differential GFP expressions when placed in different phosphate level.

Side by side comparison of construct Bba_K2447000 & BBa_K116404 where GFP/OD is expressed as a percentage. Our proposed part exhibited greater sensitivity to phosphate levels above 40uM

Our proposed phosphate construct is sensitive to phosphate concentrations from 0 to 1000 uM

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