The main goal of our 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. In principle, the exposure to high temperatures leads to an increase of the σ³² transcription factor (encoded by the rpoH gene), which subsequently enables the heat shock promoters to be recognized by the RNA polymerase. One of these promoters regulates the gene ibpA (inclusion body-associated protein A), which codes for a small heat shock chaperone. It was shown that there is a clear difference in the expression of the ibpA gene at low and at high temperatures¹. 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.

Via ligation independent cloning (LIC), we created an expression vector that contains the ibpA promoter followed by a Gfpmut3* with an LVA tag. (Figure 1) The LVA tag is a short C-terminal sequence, which was added to gfp by extension PCR. This modification results in a significantly shorter half-life than the original protein2. It can be beneficial in other experimental setups where the same bacterial culture should be able to respond to temperature several times. A longer half-life would lead to stronger and stronger signals over time. The ibpA promoter was amplified from E. coli genomic DNA. The pBID vectors are bidirectional plasmids, which would basically enable the expression of two proteins of interest from one vector. This can be useful when extending the construct with another communication pathway in the future. The plasmid contains a pUC19 origin and an ampicillin resistance gene.

To get information about the inducibility of our construct by temperature, we cultivated the bacteria under two different temperatures and measured the fluorescence. First a bacterial culture was grown overnight at 28°C. Four shake flasks were inoculated with this culture to a starting OD600 of about 0.2. Two of these cultures were then incubated at 28°C and 140 rpm, the other two at 37°C and 140 rpm. After 3 h one of the flasks, which had been previously incubated at 28°C, was incubated at 37°C and one flask, which had been previously incubated at 37°C, was then incubated at 28°C. ((Graphik??)) The other two flasks were incubated at the same temperature as before. The incubation was continued for another 3 h.  Over the 6 h total incubation time we took four samples from each flask every 15 min and measured the OD600 and the fluorescence. For detection of fluorescence we used an excitation wavelength of 485/20 nm (center value/bandpass) and an emission wavelength of 531/20 nm. The measurement was performed in a BioTek SynergyMX platereader. Each sample was measured in a fourfold determination. For blank measurements, M9-minimal media was used. The results of these measurements can be found [[https://2017.igem.org/Team:NAWI_Graz/Results|here]].

Finally, we tried a first connection of the biological system with the technical parts - the bioreactor and the robot. The bacterial culture used for the bioreactor was grown at 28°C in M9-minimal-media, diluted to a starting OD600 of 0.5 and then filled into the reactor. The setup of the experiment can be found [[https://2017.igem.org/Team:NAWI_Graz/|here]].