Part:BBa_K4044001
BphP1 (E.coli optimized)
test 123
BphP1 contain an N-terminal photosensory core domain (PCD) and a C-terminal effector domain that regulates the biological response. BphP1 has a phytochrome (PHY) motif with the chromophore biliverdin IXα (BV) covalently bound near the N-terminus. Most of the BphPs adopt inactive ground state with maximal absorption between 690 and 710 nm.
Canonical BphPs adopt an inactive ground (i.e., dark-adapted) conformation with maximal absorption between 690 and 710 nm. Upon exposure to red or short wavelength NIR light, these BphPs switch to the biologically active Pfr conformation, with maximal absorption between 750 and 760 nm. Canonical BphPs revert from Pfr to Pr in milliseconds after exposure to NIR light or in minutes to hours by thermal reversion in the dark. The bacteriophytochrome photoreceptor 1 (BphP1), found in several purple photosynthetic bacteria, senses NIR light and activates transcription of photosystem promoters by binding to and inhibiting the transcriptional repressor PpsR2.
We used truncated PpsR2 variant - QPas1 and fusing it to DNA binding domains of Gal4 from S. cerevisiae or LexA from Bacillus subtillis. DNA-binding domains not from E. coli and protein sensing NIR light opens a possibility for development of a fully orthogonal optogenetic system.
The molecular dynamics of dimers also was calculated in the OPLS-AA/L force field. The BphP1 dimers molecular dynamics values were obtained in GROMACS program. The result is reliable if Epot is negative, and on the order of 10^6-10^7 for proteins in water, depending on the system size. During the energy minimization phase, the system maximum force should not exceed 1000 kJ×mol-1×nm-1. Calculations of molecular dynamics and interaction kinetics demonstrated stability of BphP1 complex (RMSD < 0.5 nm for 100 ps, Epot = -1.6825×e+7 kJ×mol-1, Etot = -1.411×e+7 kJ×mol-1) which means the system with BphP1 dimer is stable and works correctly.
The C-terminal part of BphP1 was removed with homology modeling and the molecular dynamics of the resulting structure was calculated. As a result of analysis of the unshortened (BBa_K3032016) and shortened BphP1 sequences, it was found that the system with the unshortened BphP1 protein is more stable than with the shortened one.
We decided to change the sequence of BphP1 in presence of an already given variant (BBa_K3032016) as there was no data given if this protein could be effectively function in E.coli. 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.
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Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 760
Illegal PstI site found at 1057
Illegal PstI site found at 1504 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 760
Illegal PstI site found at 1057
Illegal PstI site found at 1504 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 246
Illegal BamHI site found at 1821 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 760
Illegal PstI site found at 1057
Illegal PstI site found at 1504 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 760
Illegal PstI site found at 1057
Illegal PstI site found at 1504
Illegal NgoMIV site found at 402
Illegal NgoMIV site found at 1153
Illegal NgoMIV site found at 1650 - 1000COMPATIBLE WITH RFC[1000]
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