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 indicated by the robot's position is passed on to a bacterial culture in a bioreactor. As a response to the pH-shift, a shift in fluorescence protein expression is received due to the pH-sensitive promoters alx and asr. This altered expression correlates with a change in color, 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.

We designed two different pH-sensitive plasmids, which are transformed in an E.coli strain. Each of the plasmids contains a pH-sensitive promoter. The promoters are induced by different pH-levels. An acidic pH leads to the expression of a red fluorescent protein (mCardinal) and an alkaline pH to a green fluorescent protein (mNeonGreen). Both fluorescent proteins contain a N-terminal TEV-site followed by a F-degron. The TEV-site allows the degradation by the TEV-protease. Once the TEV-site is cleaved off, the F-degron can be recognized by the endogenous ClpAP-machinery. This ClpAP-machinery induces the degradation of the fluorescent proteins and enables a faster change in fluorescence, when a pH-shift occurs. This addition could be beneficial in the future to avoid accumulation of fluorescence protein, if you plan to use the same bacterial culture for an extended amount of time. Therefore, the TEV-protease will be integrated into the genome by CRISPR/Cas9, a novel method for gene-editing. 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.

One of the possible applications could be a bioreactor with bacteria that control their environment via active feedback. This enables the bacteria to 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 will 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.