Part:BBa_K3515007

Troponin C, a calcium binding protein with cysteine modification(s) to bind to a biosensor and FRET

Troponin C selectively binds calcium and magnesium 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 calcium monitoring using fluorescence resonance energy transfer (FRET). Coupling this protein with two fluorophores can permit calcium detection. This composite part has coupled Troponin C with mNeonGreen in the N-terminus and mCherry in the C-terminus regions as FRET pairs. As Troponin C binds calcium 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. Special considerations must be made to the active sites of Troponin C to selectively bind calcium in its active sites and avoid magnesium binding. Calcium detection is vital as calcium is used in cells to play an important role in signal transduction pathways. Calcium is often a secondary messenger and also functions as a key neurotransmitter. Calcium is also a crucial biomarker used in clinical medicine for tracking the progression and status of patients with hypocalcemia which can lead to osteoporosis or hyperparathyroidism. As such a biosensor for calcium ion tracking may be of great interest to patients and clinicians. This part includes a mutated Troponin C to have a cysteine modification that will bind cysteine linker arms and be used for biosensor immobilization allowing the detection of calcium. 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.

Troponin C, a calcium 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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1093
  
• 1000
  COMPATIBLE WITH RFC[1000]