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Team:Uppsala-Sweden/Project
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Overall project
Main Project
Regulation of gene expression by light is a milestone in synthetic biology. This rapidly developing field has attracted lots of attention in the recent years. Light regulation introduces noninvasive, direct and advanced spatiotemporal control of engineered biological systems. The aim of this project is a continuation of developing the above mentioned regulation method.
In 2005, the world’s first light-sensing bacteria, “coliroids”, were engineered by scientists at UT Austin. Since then, as more and more naturally occurring light-sensing microorganisms are being discovered and sequenced, synthetic biologists realize there is a whole range of natural light-sensing systems at their disposal. Most of the light-sensing systems developed thus far focus on studying one light-sensing system at a time, characterizing its activation light spectra, active state, etc. There has been a lack of focus on building light-sensing systems that sense multiple wavelengths, until very recently. The ultimate objective is to introduce control of gene expression with multiple light wavelengths and demonstrate multidimensional light control as well as fine tunability of this system by making the engineered bacteria exhibit image based on three basic colors.
Side project 1: “iGERM, the motion picture”
Since biological processes are time dependent, we can take the time aspect into the bacterial picture. For instance, all of the products from light sensing will eventually degrade. A picture successfully imprinted onto a cell culture will not stay there forever. If we allowed timed control of the images by constantly changing the lighting conditions, we can make the picture change, like frames in a video camera. The ideal result is light induction of bacterial cultures ending up growing a video sequence. Hence, “pictures” become “motion pictures”.
As a side quest, the motion picture doesn’t have to be as advanced or colorful as our attempt in the main project. The motion picture could be of one color only and cartoon-like. Furthermore, since time aspect becomes crucial, there are new requirements for the photosensors and effectors. The motion picture can be expressed in other vector than the vector used in the main project, with a complete new set of photosensing system and properties. Eukaryotic phytochromes, for example, react much faster than prokaryotic photosensing systems (reference). Coupled with fast-degrading effector molecules, the expression of our “motion picture system” should be much more dynamic than the three-color sensing system.
Aim: create a photosensing system able to express images as motion pictures with fast response.
Critical: create the time delay between the light induction (“recording”) and effector response (“playing”).
Comments: no need for many colors, one is enough. The system could be radically different than the three-color-system. What about eukaryotic vectors instead of prokaryotic? The induction could even be based on protein-protein interaction and omit the interactions on transcriptional level completely. Transcription usually takes much longer time for anything to occur, something not in line with the rapidly changing motion picture.
sub project 2 : “PYP”
PYP or photoactive yellow protein is a newly discovered candidate for light-controllable synthetic systems. It’s a small protein with XXX amino acid residues, and undergoes predictable structural changes upon illumination. The N-terminus of the protein folds out from the otherwise closed conformation when illuminated, exposing the N-end residues. Recently, some research team has replaced the XXX residues from the N-terminus with GCN4, a DNA-binding domain of XXX residues. The chimeric protein XXX NAME obtained is shown to have light-induced DNA-binding activity. Basically, in the inactive state, the GCN4 end is folded into the chimeric protein, preventing its interaction with DNA. When illuminated by blue light, PYP folds out GCN4 on its N-end, allowing it to interact with DNA. GCN4 is shown to bind XXX promoters, allowing transcription of the genes downstream. It is shown however, that the chimeric protein NAME doesn’t work really well. Its active state only binds DNA twice to five times better than its inactive state. Despite its inherent drawbacks, we can still have some people working on it. The biggest incentive of developing PYP-based photosensing system is its contribution to the Registry. No PYP or its related proteins are found in the database so far. As long as we work on the genes we can submit the parts to the Registry and get credit for the work.
Aim: to experiment with PYP-based photosensing systems, design and submit the parts into the Registry.
Critical: no matter how bad the PYP-based photosystem work, we must submit something.
Comments: PYP means free credits, just because it’s very new and nobody submitted anything related to the Registry. It’s a safe card. As long as it’s played, we will win more than the value of this card. We can let the imaginations roam free on this one. Can we hang very small transcription factors on the tail of PYP? How about mutagenesis? Since we are venturing into the unknown, we won’t fail.
