[Overview]
As a functional actuator working in our ESCAPES project, we focused on 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, we attempted to construct a green-light sensing device working in E. coli via the EnvZ-OmpR signaling pathway. If photo-activated actuators responsive to different colors of light signals could be used in E. coli, we would be able to communicate with such E. coli and control them via light. Such photo-activated actuators would be put to various applications in the field of synthetic biology. To develop such photo-activated actuators, we designed a green light-activated actuator and 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.
Synechocystis sp. PCC 6803 is also known to recognize green light via chromatic acclimation sensor protein CcaS (NCBI; ABI83649) [1]. The chromophore of CcaS is assumed to be phycocyanobiline, which is the chromophore of red light sensor cph8 (BBa_I15010). The chromophore-binding domain in CcaS from Synechocystis sp. PCC 6803, as well as from phycocyanobilin-producing E. coli, showed the reversible photoconversion between a green-absorbing form (Pg λmax 535 nm) and a red absorbing form (Pr λmax 672 nm). 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 designed a fusion protein of the light responsive domains of CcaS and EnvZ.
[METHOD]
(1) Design of the green light receptor
First, we identified the sequences of the red light responsive domain (cph1) and EnvZ domain in cph8 by BLAST search. We also searched the light responsive domains in CcaS (Fig. 2). The fusion protein of the light responsive domains of CcaS and EnvZ was designed by changing the histidine kinase domain of CcaS to EnvZ domain of cph8.
(2) Construction of green light receptor (a new part)
We requested to synthesize two fragments of the photo-responsive domain of CcaS to Mr. Gene. The 1st fragment was designed containing an EcoRI and XbaI site on the N-terminal region and an NheI site at the C-terminus. The 2nd fragment was also designed containing EcoRI, XbaI and NheI sites on its N-terminal region and an NdeI site at its C-terminus. The cph8 gene, constructed by Jeff Tabor (UTAustin 2004), contained an NdeI site at the start of the EnvZ domain.
The 1st fragment was digested with EcoRI and NheI. The 2nd fragment was digested with NheI and NdeI, and the cph8 (BBa_I15010) was also digested with NdeI and PstI. All these digested samples were ligated in a single tube with a BioBrick plasmid backbone digested with EcoRI and PstI. (Fig. 3)
(3) Construction of the green light activated actuator
We tried to construct green light activated actuator composed of phycocyanobilin synthesis genes and green light receptor (Fig. 4). Unfortunately, we did not succeed to construct the entire actuator part so we could not yet confirm that it works as expected. However we do expect it to function because the amino acid sequences of the light responsive domain of CcaS and previously designed cph8 are similar and the fusion protein was constructed by simply changing the kinase domain of CcaS to EnvZ.
[Reference]
[1] Hirose et al. (2008) Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proc Natl Acad Sci U S A.105(28), 9528-33
[overview]
In a previous iGEM competition, Levskyaya et al1 designed a bacterial system that is switched between different states by red light. In this system, phycocyanobilin biosynthetic genes (BBa_I15008 and BBa_I15009) for chromophore formation and the fusion of the red-light-responsive domain with the EnvZ histidine kinase domain (BBa_I15010), which phosphorylates endogenous OmpR as the second signal, have been used as red light sensing parts (Fig. 1). In order to regulate our ESCAPE system, we tried to construct a red-light receptor using the chimeric Cph1 light receptor /EnvZ protein (Cph8)1.
The part of the photoreceptor that responds to light, phycocyanobilin, is not naturally produced in E. coli. We therefore introduced two phycocyanobilin-biosynthesis genes, ho1 (BBa_I15008) and pcyA (BBa_I15009) from Synechocystis that convert heme into phycocyanobilin8 as described previously1.
[Method]
Using standard BioBrick assembly procedures, we constructed the following red-light receptor components:
RBS-ho1-RBS-PcyA-RBS-chp8-Term,
OmpR(+) promoter-RBS
OmpR(+) promoter-RBS-GFP-Terminator
...by using the following parts:
RBS-chp8-Terminator (BBa_S03417) {APPEARS DEFECTIVE}
RBS-chp8 (BBa_S03419)
ho1 (BBa_I15008)
PcyA (BBa_I15009)
GFP-mut (BBa_E0040)
RBS (BBa_B0034)
Double Terminator (BBa_B0015).
[Results and discussion]
We constructed and confirmed the sequences of the following three parts:
Unfortunately, we were not able to confirm whether the function as expected.
We found the registered part BBa_S03417 (RBS-chp8-Terminator) to be defective by restriction mapping analysis, which showed that it does not contain the EcoRIand XbaIsites (Fig. 5). This is supported by the absence of the forward sequence in the sequence analysis data of the iGEM registry. We therefore constructed RBS-cph8-Terminator by ligating RBS-cph8 and Terminator. Next RBS-ho1-RBS-PcyA-RBS-chp8-Term was constructed by ligating [RBS-ho1-RBS-PcyA] with [RBS-cph8-Terminator].
[Reference]
1. Levskyaya et al, Nature, 438, 441-442 (2005), Engineering Escherichia coli to see light
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