Composite

Part:BBa_K4963001:Design

Designed by: Aniruddha Atul Nayak, Sumiran Kasturi   Group: iGEM23_BITSPilani-Goa-India   (2023-10-12)


Cannabinoid Receptor 1 (Modified) + INPN


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1077
    Illegal PstI site found at 1776
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1077
    Illegal PstI site found at 1776
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 333
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1077
    Illegal PstI site found at 1776
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1077
    Illegal PstI site found at 1776
    Illegal NgoMIV site found at 75
    Illegal NgoMIV site found at 408
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1575


Design Notes

The CB1 domain comprises the prominent binding sites for Honokiol. This domain functions as a holding and releasing agent for the drug. During the modification, two major factors were considered:
1. The active binding sites are retained, to allow receptor activity.
2. A sufficient fusion domain is retained, to allow formation of the composite.
The INPN part was fused to CB1 through the short linker sequence in INPN.

Source

The part is derived from two source sequences:
1. CB1- modified
Cannabinoid receptor 1 (CB1) is a part of the Endocannabinoid system, a signaling mechanism in the Central nervous system.1 It was determined to be the ideal receptor to the drug we were using in our project - honokiol, which is a STAT3 inhibitor. The receptor possesses binding sites for both honokiol and AM - 6538 (antagonist); notably in the same region.
The modified part is an optimized receptor protein with an open side for fusion, retrieved from Cannabinoid receptor 1.
2. Ice Nucleation Protein (INP - N)
The modified INP protein functions as a cell surface carrier for fused proteins. INP protein is a cell surface expression protein replete with an anchoring region (N - terminal) and a fusion domain (C - terminal).2 The INP - N protein comprises a prominent N - terminal and a new linker sequence to allow binding with other proteins.

References

1. Busquets Garcia A, Soria-Gomez E, Bellocchio L, Marsicano G. Cannabinoid receptor type-1: breaking the dogmas. F1000Res. 2016 May 24;5:F1000 Faculty Rev-990. doi: 10.12688/f1000research.8245.1. PMID: 27239293; PMCID: PMC4879932.
2. Li Q, Yan Q, Chen J, He Y, Wang J, Zhang H, Yu Z, Li L. Molecular Characterization of an Ice Nucleation Protein Variant (InaQ) from Pseudomonas syringae and the Analysis of Its Transmembrane Transport Activity in Escherichia coli. Int J Biol Sci 2012; 8(8):1097-1108. doi:10.7150/ijbs.4524. https://www.ijbs.com/v08p1097.htm