Part:BBa_K3515006

Synechococcus Phosphate Binding Protein with cysteine modification(s) and FRET to monitor phosphate

Synechococcus Phosphate Binding Protein (PiBP) selectively binds inorganic phosphate in its active site, inducing a conformation change in the N and C termini regions, respectively. This makes it a distinguishable candidate for in vivo or in vitro phosphate monitoring using fluorescence resonance energy transfer (FRET). Coupling this protein with two fluorophores can permit phosphate detection. This composite part has coupled PiBP with mNeonGreen in the N-terminus and mCherry in the C-terminus regions as FRET pairs. As PiBP binds phosphate these fluorophores in the two terminal regions come together and energy transfer occurs from donor to acceptor, permitting detection as an increase in intensity is detected. Phosphate detection is vital as phosphate is used in cells for the synthesis of DNA, phospholipids, ATP, and is involved in other signal cascades or post-transcriptional modifications. Phosphate is also a crucial biomarker used in clinical medicine for tracking the progression and status of Chronic Kidney Disease (CKD) patients. As such a biosensor for phosphate tracking may be of great interest to patients and clinicians. This part includes a mutated PiBP to have a cysteine modification that will bind cysteine linker arms and be used for biosensor immobilization allowing the detection of inorganic phosphate. The FRET pair used in this construction were considered especially for physiological detection of phosphate as they have a high intensity and are therefore able to have an expanded dynamic linear range of detection.

Phosphate binding protein fluorescent construct. Protein structures were obtained from the RCSB Protein Data Bank and a construct was made using Chimera software. Torsion angles between fluorescent and binding proteins are adjusted for display purposes. mNeonGreen (green), binding protein (blue), and mCherry (red) are all displayed using a cartoon preset in PyMOL.

Construct map displaying the entire composite parts coding region. Modifications, linkages, and fluorophore attachment points are described.

A construct map using the pSB1C3 plasmid backbone for illustration purposes.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1539
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1129
  
• 1000
  COMPATIBLE WITH RFC[1000]