Difference between revisions of "Part:BBa K3032017"
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Group: iGEM21_LMSU | Group: iGEM21_LMSU | ||
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+ | 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. | ||
+ | |||
+ | [[File:T--LMSU--ResBaseline.png|430px|thumb|left|Measurement of fluorescence intensity of YFP under Gal4-dependent promoters]] | ||
+ | [[File:T--LMSU--ResPromoters.png|430px|thumb|right|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. | 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 | + | The molecular dynamics was calculated in the OPLS-AA/L force field. 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. |
[[File:T--LMSU--Modeling-otherGal4mono-gif.gif|400px|thumb|center|Molecular dynamics simulation for Gal4-Q-PAS1 monomer]] | [[File:T--LMSU--Modeling-otherGal4mono-gif.gif|400px|thumb|center|Molecular dynamics simulation for Gal4-Q-PAS1 monomer]] | ||
[[File:T--LMSU--Modeling-RMSD-otherGal4mono.png|400px|thumb|center|RMSD for Gal4-Q-PAS1 monomer]] | [[File:T--LMSU--Modeling-RMSD-otherGal4mono.png|400px|thumb|center|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 10 | + | 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 10<sup>6</sup>-10<sup>7</sup> for proteins in water, depending on the system size. During the energy minimization phase, the system maximum force should not exceed 1000 kJ mol<sup>-1</sup> nm<sup>-1</sup>. |
− | Calculations of molecular dynamics and interaction kinetics demonstrated stability of Gal4-Q-PAS1 dimer (RMSD < 0.5 nm for 100 ps, Epot = -5. | + | Calculations of molecular dynamics and interaction kinetics demonstrated stability of Gal4-Q-PAS1 dimer (RMSD < 0.5 nm for 100 ps, Epot = -5.07×10<sup>6</sup> kJ mol<sup>-1</sup>, Etot = -4.25×10<sup>6</sup> kJ mol<sup>-1</sup>). At the same time, the BphP1-BcLOV4 lowest total score, calculated using PyDockWEB, for this model is -124.942 kJ mol<sup>-1</sup>. Total score is calculated based on electrostatics, desolvation energy and limited van der Waals contribution. |
[[File:T--LMSU--Modeling-otherGal4dim.gif|400px|thumb|center|Molecular dynamics simulation for Gal4-Q-PAS1 dimer]] | [[File:T--LMSU--Modeling-otherGal4dim.gif|400px|thumb|center|Molecular dynamics simulation for Gal4-Q-PAS1 dimer]] |
Latest revision as of 03:33, 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.
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. 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.
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.
Our findings confirm that the systems with Gal4-Q-PAS1 monomers and dimers are stable and work correctly.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 577
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]