The main goal of this experiment was to introduce a first communication pathway between robot and bacteria. Temperature regulated expression of green fluorescent protein (GFP) and the detection of the resulting fluorescence allows a simple yes or no decision. To control the expression of GFP, it is placed under the control of a temperature-sensitive promoter.

The mechanism of the heat shock response in Escherichia coli involves different genes and their corresponding promoters. On the transcriptional level, the exposure to high temperatures leads to an increase of the σ³² transcription factor (encoded by the rpoH gene), which subsequently enables many heat shock promoters to be recognized by the RNA polymerase¹. One of these promoters regulates the gene ibpA (inclusion body-associated protein A)², which codes for a small heat shock protein³. It was shown that there is a clear difference in the expression of the ibpA gene at low and at high temperatures⁴. Consequently, the sensitivity of the ibpA promoter to a temperature change can be used to create some kind of biosensor, when fused to a reporter gene.

Therefore we engineered bacteria so that an increase in temperature leads to the expression of a fluorescence protein. If the heat shock response mechanism is activated, the bacteria will produce GFP. If the bacteria are cultivated at lower temperatures, there is a significantly lower level of expression of the reporter gene. Basically, this mechanism should allow to distinguish between “active” and “inactive” by intensity of fluorescence. Due to the high background fluorescence of LB-media in the same wavelength range as GFP, the cells were cultivated in M9-minimal media for all measurements. The bacterial growth in M9-minimal-media was very slow in the beginning. Therefore the bacteria were adapted to the minimal media.

The temperature plasmid was used for a preliminary experiment in which we tried to control the robot. A video of the experiment can be found here.