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Team:Tokyo-NoKoGen/Project/photocontrol
From 2010.igem.org
Introduction
Generally, recombinant protein production in E. coli is induced by using chemical compounds. However, this way contains some problems that chemical compounds might be harmful to the environmental or the time lag for which they are took into E. coli causes controlled point of protein production difficult. In order to solve them, we focus on photoactuator which is regulation of gene expression by light. Advantages of light regulation are that we can use easily the light by simple equipment, controlled period of protein production is rapid and easy because it is start to expose E. coli to the light and there is not environmental pollution by chemical compounds. Thus, photoactuator is better protein production method to solve problems of conventional way, and would be put to various applications in the field of synthetic biology.
Why is this device needed?
Easy collection of Eco Tank containing target compound from EcoTanker is necessary in arbitrary period and place. This would be succeeded using photoactuator. The system is that it accepts particular light that is red light, green light and so on, which induced aggregation. Thus, the system easily control to concentrate EcoTanker in arbitrary period and place by only exposing E. coli to particular light.
What is this device composed inside it?
As a functional actuator working in our EcoTanker project, we focused on photoactuator, a light receptor as photo-activated actuator. Whereas the red light sensor for use as actuator had been designed and constructed by the UTAustin iGEM 2004 team [1], we attempted to construct a green light sensing device working in E. coli via the EnvZ-OmpR signaling pathway. To develop such photo-activated actuators, we had designed a green light-activated actuator and had constructed a green light receptor (BBa_K225000) which is a fusion protein of green-light responsive domains of CcaS from Synechocystis sp. PCC 6803 and EnvZ histidine kinase [2].
Synechocystis sp. PCC 6803 is also known to recognize green light via chromatic acclimation sensor protein CcaS (NCBI; ABI83649) [3]. The chromophore of CcaS is assumed to be phycocyanobiline, which is the same chromophore of red light sensor cph8 (BBa_I15010). Autophosphorylation activity of the histidine kinase domain in nearly full-length CcaS was up-regulated by pre-irradiation with green light. We focused on this chromophore-binding domain of CcaS as a green light receptor because it might work with phycocyanobilin, which can be produced in E. coli using the parts BBa_I15008 and BBa_I15009 from the Standard Registry. To develop a functional green light-activated actuator in E. coli, we tried to construct a fusion protein of the green light responsive domains of CcaS and EnvZ.
Synechocystis sp. PCC 6803 is also known to recognize green light via chromatic acclimation sensor protein CcaS (NCBI; ABI83649) [3]. The chromophore of CcaS is assumed to be phycocyanobiline, which is the same chromophore of red light sensor cph8 (BBa_I15010). Autophosphorylation activity of the histidine kinase domain in nearly full-length CcaS was up-regulated by pre-irradiation with green light. We focused on this chromophore-binding domain of CcaS as a green light receptor because it might work with phycocyanobilin, which can be produced in E. coli using the parts BBa_I15008 and BBa_I15009 from the Standard Registry. To develop a functional green light-activated actuator in E. coli, we tried to construct a fusion protein of the green light responsive domains of CcaS and EnvZ.
How does this device work in EcoTanker?
For construction of the green light activated actuator, we tried to construct green light activated actuator composed of phycocyanobilin synthesis genes, green light receptor and target green expression regulated by OmpR promoter (Fig.). To evaluate the entire green light activated actuator (BBa_2250001), it was Promoter-RBS-ho1-RBS-PcyA-RBS-GlrN-Terminater-OmpR promoter-RBS-GFP-Terminator. To expose the E. coli containing the plasmid to green light, green fluorescence of GFP would be observed.For use of green light activated actuator in EcoTanker, aggregation protein, Antigen43, coding gene expression is controlled by EnvZ-OmpR signaling pathway. This device would control aggregation of E. coli by green light irradiation.
