Part:BBa_K4044003

BcLOV4

BcLOV is a light-oxygen-voltage (LOV) flavoprotein This protein is a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet in mammalian cells [1]. This protein contains flavin as a chromophore bound to sensory domain Per-Arnt-Sim type (PAS). The migration reaction is triggered with the blue light of 450 nm where is the maximum flavin excitation in BcLOV; and reversed detaching from membrane occurs in the dark. Thus this mechanism could be useful to manageably locate specifically proteins fused with BcLOV4. It creates in this way a kind of compartmentalisation in the bacterial cell that could be used to improve the accuracy of other transcriptional regulators. We used truncated BcLOV4 variant - BcLOV4 ∆1-240 with codon optimization for E.coli. This procedure facilitate protein binding to membrane even in dim light (Fig. 1) [2]. Codon optimization of the nucleotide sequence for efficient gene expression in E.coli was performed using GENEWIZ and verified against a triplet frequency table obtained from the Codon Usage Database.

Fig. 1 BcLOV4 attached to membrane by amphipathic helix

During our project, we confirm with a computer simulation that membrane localisation of shortened BcLOV4 without N-terminal G-protein signalling (RGS) domain is mediated by a polybasic amphipathic helix located right after LOV domain. We also showed that the reversible electrostatic interaction dependent on the anionic content of the membrane without preference for a specific group. (Fig. 2).

Fig. 2 Molecular dynamics of BcLOV4 attached to dipalmitoyl phosphatidylethanolamine

Codon optimization of the nucleotide sequence for efficient gene expression in Arthrospira platensis was performed using GENEWIZ and verified against a triplet frequency table obtained from the Codon Usage Database.

1. Single-Component Optogenetic Tools for Inducible RhoA GTPase Signaling. Adv Biol (Weinh), 2021 2. Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids. Proc Natl Acad Sci USA, 2018

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 354
  Illegal PstI site found at 598
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 354
  Illegal PstI site found at 598
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Unknown
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 354
  Illegal PstI site found at 598
  
• 1000
  COMPATIBLE WITH RFC[1000]