Part:BBa_K3515008
Potassium binding protein with cysteine modification(s) to bind to a biosensor and FRET to monitor p
Potassium binding protein (Kbp) selectively binds potassium 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 potassium monitoring using fluorescence resonance energy transfer (FRET). Coupling this protein with two fluorophores can permit potassium detection. This composite part has coupled Kbp with mNeonGreen in the N-terminus and mCherry in the C-terminus regions as FRET pairs. As Kbp binds potassium 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. Potassium detection is vital as potassium is used in cells to play an important role in fluid and electrolyte homeostasis, which is crucial for functioning of the neurological, cardiovascular, and immune system. Potassium is also a crucial biomarker used in clinical medicine for tracking the progression and status of patients with hypo- or hyperkalemia, which may provide insights into Chronic Kidney Disease (CKD) or diabetic ketoacidosis. As such a biosensor for potassium tracking may be of great interest to patients and clinicians. This part includes a mutated Kbp to have a cysteine modification that will bind cysteine linker arms and be used for biosensor immobilization allowing the detection of potassium. 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.
Potassium 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 766
Illegal AgeI site found at 823
Illegal AgeI site found at 883 - 1000COMPATIBLE WITH RFC[1000]
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