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 there is blue light offered to this system, YF1 will be dephosphorylated and it will result in the dephosphorylation of FixJ, thus preventing the FixK2 promoter. Then, gene expression level will be greatly reduced. Some previous testing results can be seen here and the overall system testing can be seen here.

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 find all the parametres indicate this system is suitable for us to use in our designed box. 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 Element Regulated

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 key enzyme in the fatty acid synthesis pathway, FadD, which will prevent fatty acid synthesis. After detecting the fatty acid amount in the culture and inside the cell and comparing different total fatty acid amount under different light intensities, we are able to prove this blue-light controlled CRISPRi system can be used to regulate genomic genes thus regulating the metabolism in vivo. Moreover, this will be one example showing our system is versitile and universal.

To test the whole system, the blue light system controlled sgRNA is designed to target one of the fatty acid synthetic enzymes, FabZ. Together with TesA and FabI controlled by red and green lights, 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 the whole system can optimize metabolic pathways via regulating genomic genes, which is our final goal.

Design

The plasmid we get at last is as follows:

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.

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 are very happy about this little story. First 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; Second is that we help discover a mistake in a published research incidentally, which will help other researchers use this light system more correctly.

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.