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Team:Exeter/Project
From 2013.igem.org
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Overall project
Our overarching aim is to further improve upon an area that many past iGEM teams have studied; using multi-wavelength light stimulus to generate variable outputs. We are especially inspired by Uppsala’s 2011 project, and the “Hello World” bio-film from UT Austin in 2004. Additionally we aim to produce a standardized brick to be twinned through a series of NOT gates with the available well characterized light input machinery, enabling implementation of any trio of outputs. Acute control over each output should be possible by varying wavelength input. To show this off we hope to use CMY colour wheel as our output (as opposed to the previously studied RBY), to produce a bio-photograph. We also hope to use non-fluorescing proteins as our pigments, as we want a picture that simply develops, visualisable without any further stimulation/excitation.
Although this has been a subject many teams have grappled with in the past (with differing degrees of success), we are confident that we can add to the mass of data and introduce new BioBricks related to light-sensing and output in the iGEM database, and hopefully go several steps further towards making full-colour photographs using bacteria.
Considering modelling, we hope to analyse the absorption spectrum of each pigment, both alone and in combination. Visualisation of light sensitivity throughout the system will enable calibration of the light input ensuring a reliable and sensical output. Further to this other teams reported their light sensing systems working better at temperatures <37 degrees, which we will also investigate.
As a sideline, we’re also going to test a new way of preserving and presenting bacterial culture using pouring plastics. Unfortunately, this would kill the bacteria, but should give academics a new way to physically present colonies and cultures they have been working on. For us, it will allow a way of easily transporting our “photographs” without unwieldy plates and gels.
As mentioned we will be using additive and subtractive colour combinations to allow synthesis of the correct pigment output, corresponding to which colours of light they are exposed to. For example the NOT gated input of red light represses synthesis of cyan pigment enabling an output of yellow and magenta pigment.
Ensuring reliable activity across NOT gates in our pathways is key in our project. For the blue and red light sensors, the presence of their corresponding wavelength of light causes autophosphorylation of an intermediate protein. Phosphorylated intermediates then freely bind as repressors to the corresponding output gene (cyan for red light, yellow for blue light). With the green light sensor, a signal inverter will be introduced, as the FixJ system acts as an activator not a repressor (the exact opposite of what we want). Instead of acting on the gene coding for the output (magenta) the phosphorylated intermediate binds instead to a gene coding for the cI protein used in the lambda phage (a bacteriophage which infects E. coli). Once synthesised the cl protein acts as the output (magenta) repressor.
