The aim of our project was to create a robot-bacteria interface, where information processing is done by a bacterial culture that exhibits a feedback loop with a mobile robot. To enable communication between bacteria and robot, we decided to engineer E. coli cells to respond to changes in pH with a change in fluorescence. To achieve pH-dependent expression of fluorescence protein, we used two different promoters: the acid-inducible asr promoter and the alkaline inducible alx promoter.

An important area at the cutting edge of technology is the integration of biological systems in mechanical and automated systems. For our iGEM project we therefore chose to take a new approach on the integration of biology into technology. Our goal is the creation of a bacteria computer interface that acts in a constant feedback loop. We create a pH sensitive E. coli strain that expresses detectable proteins according to a pH level. The pH level is determined by the position of a mobile robot in an arena with a virtual pH gradient. The pH level of the robot's position is passed on to bacteria that grow in a bioreactor, as a response to the pH shift, a shift in protein expression is made due to pH sensitive promoters. This altered expression correlates with a change in color, due to a change in expression, that is detectable with an optical system. The data from the optical signal is then send back to the robot resulting in a response in form of a directional change. Thus, we can control the motion of the robot through the arena with a constant feedback loop to the bioreactor. Furthermore, it is planned, to characterize and develop one of the used pH sensitive promotors, as a Biobrick.

It is planned to design a pH-sensitive E. coli strain with two different plasmids. Each of the plasmids contains a pH sensitive promotor. The promotors are induced by different pH levels. An acidic pH leads to the expression of a red fluorecent protein and an alkaline pH to a green fluorecent protein. Both fluorecent proteins contain a N-terminal TEV-site followed by a F-degron. The TEV-site, which allows the degradation by the TEV-protease. The F-degron should increase the velocity of the degradation of the fluorecent protein by recruiting endogenous ClpAP machinery. This is important to provide a faster color change due to pH shift. The TEV-protease is integrated into the genome by CRISPR/Cas9. Due to the assay, it is necessary that the proteins are produced and degraded very fast. Therefor, the protein exhibits a quick maturation time. Based on the combination of the TEV-protease and a quick maturation time, it is possible to gain rather quick changes of the fluorescence in the media. The fluorescent signals are detected by a camera, leading to maneuvering the robot through an arena. Depending on the fluorescent color of the media, the robot turns either left or right. For a better performance of transcription and expression the acid-inducable asr-promotor should be characterized and further developed. Therefor, it is planned to mutate some regions, like the RBS (ribosome binding site) of the promotor to optimized it.

One of the possible applications could be a bioreactor with bacteria that control their environment via active feedback. Meaning that the bacterias give a signal, if they are in need for a different media composition, for example if more nutrients or a switch of the pH level become necessary bacteria indicate it and a robot automatically adapts the medium to the circumstances. The technology also holds potential in the creation of reusable biosensors or a controlled bioremediation effort, where bacteria are mobile due to the connection to a robot host.