Difference between revisions of "Part:BBa K3032017"

Revision as of 01:47, 22 October 2021

Gal4_Q-PAS1 repressor

Gal4_Q-PAS1 repressor is a fusion protein which consists of Gal4 domain (from Saccharomyces cerevisiae) and Q-PAS1 (from Rhodopseudomonas palustris). This repressor binds to promoter containing Gal4 DNA binding sequence and dissociates when it is illuminated with NIR light. Bphp1 (BBa_K3032016) and ho1 (BBa_K3032015) proteins required for system to work.

Contribution

Group: iGEM21_LMSU

We tested both base activity of these promoters and the inhibition efficiency of Gal4 with them to get a proper characterisation of these parts. The baseline activity was tested by YFP output with strong RBS used (BBa_J34801). We compared the relative activity of modified promoters with the original J23119 promoter. As a negative control also, original promoter was taken. Thus we get two new promoters dependant on Gal4 that can be used in procaryotic organisms that we consider to be a significant part improvement. Our further step is to try other Anderson promoters to create a library of bacterial Gal4 dependant promoters that there could be more chance to select the best fitting element for different chassies.

Measurement of fluorescence intensity of YFP under Gal4-dependent promoters

Observed inhibited relative activity of Gal4-dependent promoters in presence of QPAS1-Gal4 protein

We performed the molecular dynamics simulation for Gal4-Q-PAS1 chimeric protein, since there was no data on the stability of the system with Gal4-Q-PAS1 monomers and dimers.

The molecular dynamics was calculated in the OPLS-AA/L force field by LMSU team because LMSU team created an optogenetic system based on this protein and optimized the Gal4-Q-PAS1 monomers and dimers. Once we were sure that the obtained structures deviate insignificantly when calculating the molecular dynamics in water (RMSD < 0.5 nm for 100 ps), and the monomers remain stable, we proceeded to construct dimers.

Molecular dynamics simulation for Gal4-Q-PAS1 monomer

RMSD for Gal4-Q-PAS1 monomer

The Gal4-Q-PAS1 dimers molecular dynamics values were obtained in GROMACS program. The result is reliable if Epot is negative, and on the order of 10⁶-10⁷ 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 Gal4-Q-PAS1 dimer (RMSD < 0.5 nm for 100 ps, Epot = -5.07×10⁶ kJ mol^-1, Etot = -4.25×10⁶ kJ mol^-1). At the same time, the BphP1-BcLOV4 lowest total score, calculated using PyDockWEB, for this model is -124.942 kJ mol^-1. Total score is calculated based on electrostatics, desolvation energy and limited van der Waals contribution.

Molecular dynamics simulation for Gal4-Q-PAS1 dimer

RMSD for Gal4-Q-PAS1 dimer

The system's potential energy for Gal4-Q-PAS1 dimer

The system's total energy for Gal4-Q-PAS1 dimer

Our findings confirm that the systems with Gal4-Q-PAS1 monomers and dimers are stable and work correctly.

Sequence and Features