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After designing red and green light controlled gene expression systems, we hope to find another one that is regulated by blue light in order to broaden the range of the application of our systems, meanwhile to avoid the overlapping effect among lights with different wavelengths.
Fortunately, we find out that there is a perfect candidate in projects of 2011 Uppsala Team. With further paper-searching work based on its reference page, we find an interesting published paper named "From Dusk Till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression", which also provides us with lots of useful information. Therefore, we decide to select this one as our third light-controlled system.
Blue Light Sensing System
There are three parts in this blue light regulated gene expression system in total. First, YF1 protein. The histidine kinase YF1 protein is a fussion protein that employs a light-oxygen-voltage blue-light photosensor domain. Second, FixJ protein. This is a regulator and its status can be controlled by YF1. Third, FixK2 promoter. This promoter is regulated by FixJ.
Suppose that there is no light, or say, in the absence of blue light, YF1 will phosphorylate its cognate response regulator FixJ, which then drives robust gene expression from the FixK2 promoter. However, if blue light is introduced to this system, YF1 will be dephosphorylated, which will result in the dephosphorylation of FixJ, thus preventing the FixK2 promoter. Then, gene expression level will be greatly reduced.
The wavelength of blue light used in this system is 470nm, and it has a minimal overlapping effect with red and green light. There is also a figure in this published paper showing different gene expression levels with different light intensities. More importantly, after careful calculation we found all the parameters indicated that this system was suitable for us to use. Another interesting and important but unexpected brief interlude happened during our calculation, and this will be mentioned at the "Others" part in this page.
Downstream Regulation
We have already got the FixK2 promoter, and the next thing is to choose suitable reporter genes and functional genes and put them downstream of this promoter.
To test this system qualitatively, the sgRNA targeting to RFP is regulated by FixK2 promoter. Considering the mechanism of CRISPRi system, in the absence of blue light(wavelength=470nm), RFP will be knocked down and under the blue light RFP will be expressed normally.
To test this system quantitatively, the sgRNA targeting to luciferase is regulated by FixK2 promoter. Then we change light intensity and detect gene expression amount. Since the amount of expressed luciferase can be tested indirectly by chemiluminescence detector, it is likely that at last we could get the relationship between light intensity and gene expression amount.
To test this system practically, the sgRNA targeting to one of the fatty acid synthetic enzymes, FabZ. Together with TesA and FabI, we hope to maximize the producing efficiency of the fatty acid synthetic pathway by changing the ratio of these three enzymes amount. From this experiment, we will prove that our system can optimize metabolic pathways via regulating genomic genes.
Sensor Design
The plasmid we get at last is as follows:
 Fig.1 Plasmid Construction: Final version of Blue light sensing system The whole construction process follows the same way. We get the main part from part kit from iGEM and clone it into the constant expressing operon designed by ourselves, and then the whole operon is cloned into a pCDFDuet vector.
After a long long constructing process, up till now all of our plasmids have been successfully constructed and we are ready to test their functions next.
Others
During the designing process, we calculated that the light intensity needed to stimulate blue light controlled gene expression system was too small. Wondering the feasibility of this system, we contacted the corespondent author of the reference paper. Soon we got the reply, and unexpectedly, the author realized the published paper had a small mistake. The unit in that paper was wrong, it should be 1000 times larger. He also said that they would contact the journal and submit an erratum to the journal in due course.
Looking back, we were very happy about this little story. For one thing, we can use this system since after another round of calculation, the result shows that it is a pretty good choice to use this system; the other thing is that we helped discover a mistake in a published research incidentally, which will help other researchers use this light system more accurately.
Reference
OHLENDORF, R., VIDAVSKI, R. R., ELDAR, A., MOFFAT, K. & M GLICH, A. 2012. From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. Journal of Molecular Biology, 416, 534-542.
M GLICH, A., AYERS, R. A. & MOFFAT, K. 2009. Design and Signaling Mechanism of Light-Regulated Histidine Kinases. Journal of Molecular Biology, 385, 1433-1444.
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