Part:BBa_K5431014
FU1 (UirS-UirR)
Introduction
The UV intensity response sensor (UirS) and regulator (UirR), make up a two-component regulatory system. This type of system is known to consist of the sensor histidine kinase and response regulator, along with an output promoter (Ramakrishnan & Tabor, 2016). The two component system allows the cyanobacteria it originates in to respond to certain wavelengths of light. This photoreceptor, also known as a cyanobacteriochrome (CBCR), is activated at 400 nm and deactivated at 530 nm. The sensor and regulator are both bound to a membrane and when exposed to the appropriate wavelength, the sensor phosphorylates the regulator. A chromophore is also a necessary component to enable light absorption of the system. The proper wavelength absorption induces isomerization of the chromophore, turning the CBCR into an activated state. On the other hand, the absorption of the reversion wavelength will bring it back to the ground state. When UirS phosphorylates UirR, transcription is activated from the promoter (PcsiR1-109) towards the gene of interest.
The genes ho1 (heme oxygenase) and PcyA (Phycocyanobilin ferredoxin oxidoreductase) together form PCB, otherwise known as a chromophore. A chromophore is essential for the absorption of light in photoreceptors found in cyanobacteria, called cyanobacteriochromes. The ho1 and PcyA work in conjunction in a system to produce PCB. In this system, the heme yields BV IXa, Fe^2+, and CO. PcyA then takes the BV IXa product and reduces it to make PCB (Okada, 2009). A RBS is included between both proteins in order to aid higher production and mRNA stability.
The UV intensity response sensor (UirS), regulator (UirR), chromophore (ho1 and pcyA), and gene output (sfGFP) make up the composite part FU1. This cycle, we introduce FU1 as a novel optogenetic device for protein engineering in PACE. With FU1, teams can now monitor gene expression more affordably and with greater accuracy/control than chemical inducers.
Modeling
We have done experiments with FU1 to prove that it works as expected. We found there is high gene expression in UV violet light. Lower gene expression in darkness. Lowest gene expression in green light. Thus, UV violet light (400 nm) activates the system, while darkness and green light (530 nm) have similar effects of deactivating the system.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2870
Illegal EcoRI site found at 3812
Illegal XbaI site found at 426
Illegal XbaI site found at 1803
Illegal XbaI site found at 3022
Illegal SpeI site found at 53 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2870
Illegal EcoRI site found at 3812
Illegal NheI site found at 3799
Illegal SpeI site found at 53 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2870
Illegal EcoRI site found at 3812
Illegal BglII site found at 611
Illegal BglII site found at 1800
Illegal BglII site found at 2655 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2870
Illegal EcoRI site found at 3812
Illegal XbaI site found at 426
Illegal XbaI site found at 1803
Illegal XbaI site found at 3022
Illegal SpeI site found at 53 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2870
Illegal EcoRI site found at 3812
Illegal XbaI site found at 426
Illegal XbaI site found at 1803
Illegal XbaI site found at 3022
Illegal SpeI site found at 53
Illegal NgoMIV site found at 118
Illegal NgoMIV site found at 4911 - 1000COMPATIBLE WITH RFC[1000]
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