Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Maria Mollerup
Date created: 2010-10-15 11:00:00
Date modified: 2015-05-08 01:12:08
Photosensor generator
| Types | DnaRegion |
| Roles | Generator engineered_region |
| Sequences | BBa_K343007_sequence (Version 1) |
Description
Molecular mechanism of the photosensorThe fusion,chimera-protein coupled to the chemotaxis pathway. Figure taken from Trivedi et al.(2)
The fusion,chimera-protein coupled to the chemotaxis pathway. Figure taken from Trivedi et al.(2)
The photosensor acts directly on the tumbling frequency by effecting E. Coli's normal chemotaxis pathway. When exposed to bluelight, the sensory rhodopsin II will absorb the photons and undergo a change in ultrastructure, which is being transduced through HtrII on to the Tar domain. This effects decreases the autophosphorylation of the CheA protein, which in turn again decreases the amount of phosphorylated CheY, which just means that less of it will get phosphorylated. If there is less of the CheY-P, then there is a smaller chance of one of these molecules binding to the flagellar motor and making it turn clockwise, thereby inducing a lowered tumbling frequency in the system. The photosensor can also act in the opposite way, inducing a higher tumbling rate in the bacteria. Which effect the sensor will have depends on where NpHtrII and StTar are fused in the HAMP domain. If the fusion contains 20 more basepair of the HtrII domain and 20 less of the Tar domain, the photosensor would have the opposite effect and would be increasing the autophosphorylation of CheA. Usage and parameters
The part requires retinal to work in E.Coli. This can be achieved through adding retinal to the liquid growth medium and/or the plates. Currently we are doing experiments on wether the part also functions with an internal retinal source, ie retinal synthesis in E. Coli. On top of adding retinal the cells will have to be grown in the dark for at least two hours after the addition of retinal to the growth medium if you want to see an effect. This is so that the photosensor is not exposed to light before the experiments and will result in maximum output if exposed to blue light. Results
Bacteria containing this part will exhibit a lowered tumbling rate when exposed to blue light (wavelengths around 350nm - 450nm). This was analysed with the help of video microscopy and the open source software "CellTrack". The individual cells trajectory was tracked and their speed measured. The tracking results are as follows:
From left to right, trajectory of: E.Coli with photosensor exposed to blue light, E.Coli with photosensor exposed to red light and E.Coli Mg1655 Wildtype exposed to blue light: (Blue dots show the location of the cell in the given frame, so the number of dots equals the number of frames from the sample.)
The phototaxic bacteria move more in a straight line when exposed to bluelight, as can be seen when comparing the trajectories of the thee bacteria given earlier. These were taken from a batch of 10 cells tracked per sample.
Notes
Design NotesNpSopII-NpHtrII-StTar fusion-chimera protein sequence from Jung K-H, Spudich EN, Trivedi VD and Spudich JL (1).
The first 224 amino acid residues come from the NpSopII gene, encoding a bluelight photon receptor with 15 residues removed at the C-terminal end. The following 9 amino acids are a linker. the last part is HtrII fused with Tsr from E.Coli at the HAMP domain. The complex' first 125 amino acid residues come from HtrII and the remaining 279 from Tsr.
The constitutive promoter R0040 was chosen for its medium strength, so that there would be a rather large amount of the fusion, chimera protein expressed, but not so much that it would be lethal to the cell. The easy repression and inhibition of repression through TetR and tetracycline makes for an easily controlled expression if that is needed. The doible terminator B0015 was chosen for it's reliability and good reviews on the partsregistry.
Source
SourceSensory Rhodopsin II and HtrII domains were taken from the genome of Natronomonas Pharaonis. The Tar methyl accepting chemotaxis protein was taken from Salmonella Enterica Serovar Typhimurium. The synthetic linker and fusing of the HAMP domains of HtrII and StTar was done by the team behind the referenced article (1).
| Access | Instance | Definition |
| public public public | BBa_J13002 BBa_K343003 BBa_B0015 | BBa_J13002 PS BBa_B0015 |
| Sequence Annotation | Location | Component / Role(s) |
| BBa_J13002 BBa_K343003 BBa_B0015 | 1,74 81,2045 2054,2182 | BBa_J13002 PS BBa_B0015 |