Project
Project overview
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.
Side project 2 : “i, PYP”
PYP or photoactive yellow protein is a newly discovered candidate for light-controllable synthetic systems. It’s a small protein of 125 amino acid residues. It 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 25 amino acid residues from the N-terminus with GCN4, a basic leucine zipper (bZIP)-type DNA-binding protein. The chimeric protein GCN4Δ25PYP-v2 obtained is shown to have light-induced DNA-binding activity. Basically, in the inactive state, the GCN4 end is sequestered when being tightly packed against the PYP domain, preventing its interaction with DNA. When illuminated by blue light, GCN4Δ25PYP-v2 folds out GCN4 on its N-end, allowing the GCN4 domain to interact with DNA. GCN4 is shown to bind AP1 promoters, thus allowing transcription of the genes downstream.
It is shown however, that the chimeric protein GCN4Δ25PYP-v2 is in need of improvement. Its active state only binds DNA twice to five times better than its inactive state. Despite its inherent drawbacks, we see potential in this light-sensing protein, partly due to its mechanism. The way GCN4Δ25PYP-v2 works is through allosterism, which have been anticipated as the best approach of constructing light-sensors that do not require e.g. dimerization, thus reducing cross-talk and overall noise of the system. Therefore, our iGEM team will try to build and test photosensing systems based on GCN4Δ25PYP-v2, as well as submitting the DNA as BioBrick to allow further investigation for other iGEM teams in the future.
Project Details
Part 2
The Experiments
Part 3
== Results ==
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