Part:BBa_K3515009

Glucose binding protein with cysteine modification(s) to bind to a biosensor and FRET to monitor glu

Glucose/Galactose binding protein (GGBP) selectively binds glucose 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 glucose monitoring using fluorescence resonance energy transfer (FRET). Coupling this protein with two fluorophores can permit glucose detection. This composite part has coupled GGBP with mNeonGreen in the N-terminus and mCherry in the C-terminus regions as FRET pairs. As GGBP binds glucose these fluorophores in the two terminal regions move apart from another and energy transfer dissipates from donor to acceptor, permitting detection as a decrease in intensity is detected. Glucose detection is vital as glucose is used in cells to play an important role in critical metabolic pathways, which is crucial for maintaining energy homeostasis. Glucose is also a crucial biomarker used in clinical medicine for tracking the progression and status of patients with diabetes mellitus which may provide insights into pancreatic functioning and metabolic state. As such a biosensor for glucose tracking may be of great interest to patients and clinicians. This part includes a mutated GGBP to have a cysteine modification that will bind cysteine linker arms and be used for biosensor immobilization allowing the detection of glucose. 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

Glucose/Galactose 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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]