The Caltech iGEM outreach initiative turned into an extensive collaboration with California Institute of the Arts to create animations from fluorescent bacteria. Previous studies have shown that high-definition images can be produced by Escherichia coli using projection of a pattern of light onto the bacteria. The principal objective of our collaboration was to introduce a degradation tag that would degrade the emission of red fluorescent protein (mCherry) while it is created and use a projection of moving images to create an animated bacterial lawn.

Originally, bacterial photography was performed in a special knockout E. coli strain RU1012. In a regular E. coli strain, the system uses a membrane-bound sensor (EnvZ) that is sensitive to osmotic shock. In RU1012, the EnvZ sensor is “knocked out” and replaced with a Cph8 light sensitive sensor. The system is a two-part system comprised of a membrane-bound extracellular sensor (Cph8 light receptor) that responds to light and an intracellular response regulator (EnvZ-OmpZ). The cofactor, Phycocyanobilin (PCB), must also be present for the Cph8 light receptor to function and is converted with the help of two biosynthesis genes: ho1 and pcyA4.

When the projection of light is shone on the E. coli, the EnvZ-OmpZ responds and phosphorylates the OmpR promoter, which turns on lacZ and generates a black output. The differing amounts of black output created provide the contrast for a photograph. This system allows a lawn of bacteria to function as a biological film, such that when a projection of a specific image of light is shone on the bacteria, a highly detailed image is produced4. The sheer number of bacteria in one square inch of a petri plate creates an incredible resolution for photography as well as animation.

The main goal of this project was to use the system for bacterial photography and replace lacZ (black permanent output), for a temporary red output with a derivative of red fluorescent protein, mCherry, and a degradation tag that varies in the last three amino acid sequence (LVA, AAV). The degradation tag continuously degraded the red fluorescent protein (RFP) produced by the mCherry in order to create a temporary palette, as opposed to the permanent bacterial Polaroid photograph. The LVA degradation tag has a longer half-life than the AAV degradation tag3. When the system is constructed and a lawn of E. coli is growing on a petri plate, a green filter is placed over the LED projector lense, cutting off all the white light. The green light then passes through the petri plate and excites the red fluorescent protein. A red filter is then placed over the lenses of a video camera that is pointed towards the petri plate, so that only the fluorescence of the RFP is seen. This setup allows only the desired fluorescent animation to be captured.