Team:NAWI Graz/TemperaturePlasmid

TEMPERATURE PLASMID

Our temperature sensing construct is based on a promoter which is activated by the heat shock response of Escherichia coli. A rise in temperature leads to increased production of green fluorescent protein and a resulting increase in fluorescence. This mechanism allows to distinguish between induced and uninduced. When used as a communication pathway, it results in a simple yes / no decision.

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.


References
1. Nonaka, G., Blankschien, M., Herman, C., Gross, C. a & Rhodius, V. a. Regulon and promoter analysis of the. Genes Dev. 20, 1776–1789 (2006).
2. Allen, S., Polazzi, J., Gierse, J. & Easton, A. Two novel heat shock genes encoding proteins produced in response to heterologous protein expression in Escherichia coli. J Bacteriol 174, 6938–6947 (1992).
3. Gaubig, L. C., Waldminghaus, T. & Narberhaus, F. Multiple layers of control govern expression of the Escherichia coli ibpAB heat-shock operon. Microbiology 157, 66–76 (2011).
4. Rodrigues, J. L., Sousa, M., Prather, K. L. J., Kluskens, L. D. & Rodrigues, L. R. Selection of Escherichia coli heat shock promoters toward their application as stress probes. J. Biotechnol. 188, 61–71 (2014).