Part:BBa_K4444017

nTEV-iLID-mCherry

Introduction

nTEV iLID mCherry is a modification of the FRB-nTEV system designed by Katja Leben from ​​Slovenia in 2016 (BBa_K1965039). We improved this part by making it optogenetic and adding a fluorescent protein (mCherry)(1). This part is meant to be used in conjunction with cTEV-sspb-BFP (BBa_K4444018).

Design of nTEV-iLID System

Optogenetics

Optogenetics is a powerful research tool for controlling cell behavior that offers precise spatiotemporal control by varying the wavelength of lights used. The spatial precision can be achieved at the level of micrometers while the temporal precision can be achieved at <1ms (Zhu et al.). This light input generates a response in photosensitive proteins bound to target proteins, which allows researchers to control different aspects of cell activity.

iLID-sspb System

iLID (improved light-induced dimer) and sspb (stringent starvation protein B) are a pair of heterodimerizing proteins that reversibly bind under 450 nm light within seconds of exposure. iLID consists of a bacterial SsrA (small stable RNA A) peptide embedded in the naturally occurring photoswitch AsLOV-2 (Avena sativa light-oxygen voltage 2 domain) (Guntas et al.). Before light activation the SsrA is sterically blocked from its binding partner sspb by AsLOV-2 domain however upon light exposure the domain undocks and heterodimerization takes place. Optogenetic heterodimerizing proteins are very useful in biological signaling studies because they allow cellular localization of POIs. For example iLID has already been applied in reversible controlling small GTPase signaling, which are enzymes that regulate cytoskeletal reorganization, cell polarity, cell cycle progression, gene expression and many other significant events in cells, such as the interaction with foreign particles (Song et al.).

Mechanism

This part contains an ER localization tag which keeps the system embedded in the ER membrane. Under photoactivation, the cTEV-sspb system heterodimerizes with the nTEV-iLID system. Under the microscope, one should expect to see cytoplasmic BFP (cTEV system contains BFP) and mCherry in the ER. When blue light is shined, BFP should be recruited to the ER membrane as iLID and sspb dimerize.

References

[1] Zhu, Danqing, et al. “Optogenetic Application to Investigating Cell Behavior and Neurological Disease.” Frontiers in Cellular Neuroscience, vol. 16, 22 Feb. 2022, 10.3389/fncel.2022.811493. Accessed 16 Aug. 2022.

[2] Guntas, Gurkan, et al. “Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins.” PNAS, vol. 112, no. 1, 2014, pp. 112-117, https://doi.org/10.1073/pnas.1417910112.

[3] Song, Siyang, et al. “Small GTPases: Structure, Biological Function and Its Interaction with Nanoparticles.” Asian Journal of Pharmaceutical Sciences, vol. 14, no. 1, Jan. 2019, pp. 30–39, www.sciencedirect.com/science/article/pii/S1818087618304641, 10.1016/j.ajps.2018.06.004.

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 100
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1258
  
• 1000
  COMPATIBLE WITH RFC[1000]