Part:BBa_K5466030

Split mCerulean C-terminal. Saccharomyces cerevisiae codon optimized

C-terminal part of the split mCerulean (residues 155-238) with a flexible linker. It can re-associate with split mCerulean N-terminal (BBa_K5466029) in vivo and form mCerulean that can emit fluorescence. Use to study protein–protein interactions.

Can be fused with EpoR-D2-TM (BBa_K5466007) to verify that ligand-induced dimerization is produced on the cytoplasmic membrane by the two ligand-binding domains targeting the same ligand to test if they are good candidates as LBD on the GEMS platform or Yeast Patrol.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 84
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

mCerulean

mCerulean is cyan fluorescent protein that can be use in vitro and in living cells, engineered from ECFP.

mCerulean originated from ECFP, which, despite several spectroscopic disadvantages such as a low quantum yield, low extinction coefficient, and a fluorescence lifetime best described by a double exponential, was commonly used. To improve ECFP's properties for FRET measurements, a site-directed mutagenesis approach was employed by Rizzo et al. (2004) to address these limitations. The resulting variant, named Cerulean by Rizzo et al. (2004) (ECFP/S72A/Y145A/H148D), exhibited a significantly improved quantum yield, higher extinction coefficient, and a fluorescence lifetime best fit by a single exponential. Cerulean is 2.5 times brighter than ECFP.

BiFC

Studying protein–protein interactions (PPIs) is essential for understanding the molecular mechanisms that drive protein function. These interactions can vary between cells, occurring in different locations and with varying strengths. To fully grasp protein function, it’s crucial to capture PPIs in their natural cellular context and conditions. Bimolecular fluorescence complementation (BiFC) facilitates the study of PPIs in diverse native contexts and has proven effective in various cell types, from bacteria to mammals. This technique relies on the ability of monomeric fluorescent proteins to reassemble from two separate fragments when in close proximity. When candidate proteins are fused to these complementary fragments, a fluorescent signal is generated upon interaction, enabling the visualization of weak and transient PPIs. BiFC can also be used to investigate multiple PPIs simultaneously using a multicolor approach.

In addition to detecting PPIs, BiFC provides insights into the subcellular localization and affinity of these interactions in living cells. However, BiFC is irreversible, which, unlike FRET, prevents the analysis of dynamic complex formation and dissociation. Nonetheless, BiFC enhances the detection of weak PPIs by stabilizing them, even at normal expression levels. It can also be applied to investigate distinct PPIs simultaneously using a multicolor setup. This is achieved by complementing N-mVenus with C-mCerulean, which has a different excitation and emission wavelength than mVenus and mCerulean.

Since the Patrol Yeast signaling platform also uses a system originally developed to study PPIs and is based on split-ubiquitin, we thought that BiFC was an ideal technique, due to its similar mechanisms, to reliably confirm that the LBDs intended for use in the signaling platform produced ligand-induced dimerization.

References

Jia, Y., Bleicher, F., Merabet, S., & Reboulet, J. (2021). Bimolecular Fluorescence Complementation (BiFC) and Multiplexed Imaging of Protein–Protein Interactions in Human Living Cells. Methods In Molecular Biology, 173-190. https://doi.org/10.1007/978-1-0716-1593-5_12

Rizzo, M. A., Springer, G. H., Granada, B., & Piston, D. W. (2004). An improved cyan fluorescent protein variant useful for FRET. Nature Biotechnology, 22(4), 445–449. https://doi.org/10.1038/nbt945

Weber-Boyvat, M., Li, S., Skarp, K., Olkkonen, V. M., Yan, D., & Jäntti, J. (2014). Bimolecular Fluorescence Complementation (BiFC) Technique in Yeast Saccharomyces cerevisiae and Mammalian Cells. Methods In Molecular Biology, 277-288. https://doi.org/10.1007/978-1-4939-2309-0_20