Lysis System - dpB.014 |
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To avoid a ion interference with the other redox-based pathway we used for the input, we preferred to use a lysis system to connect electrically the output of our circuit. The idea was to view the number of cells in the medium as a resistance value of the medium, allowing us to measure it using a potensiostat, thus connecting the output our circuit with an electrically measurable unit. As shown in [1], growing bacteria’s capture Ions present in their media, and, in doing so, decrease the electrical conductivity of the medium. |
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In a previous experiment [2], we showed that our custom IO Rodeo potentiostat was capable of capturing Electrical conductivity of the medium as a proxy for cell density. To use this property as an output of our system, we designed an Output Plasmid expressing lysis protein under the control of an inducible promoters and characterised its ability to modulate the final steady state OD of the growing population as a function of the concentration of inducers. |
Materials and methods |
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E. coli wild Marionette strain [3] transformed with the dpB0.14 composite part (pLux-C86m (φX174 Lysis Protein (X174E))) was grown overnight in LB+ Kanamycin. In the morning, cultures were dilluted to a concentration of 1:100 in fresh media in 96 Well place. |
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Dose response experiment of varying concentration of OC6 (from 400 uM to 0 uM)
was conducted on the Tecan Infinite® 200 PRO plate reader.
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Results |
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Figure 1 show that varying concentration of OC6 yielded a range of final OD600 from 1.28 (for no inducer) to no growth for higher inducer values. Importantly, variation of concentration in the range of from 1uM to 100uM yielded an wide range of final OD compatible with our Electrical conductivity measurement conducted in [2]. Taken together, these results show that varied induction of the lysis system yielded a wide range of electrically readable signals and therefore validate its functioning as an output system for our electro-genetic toolkit. |
References |
1. Din, M.O. *et al.* (2020) ‘Interfacing gene circuits with microelectronics through engineered population dynamics’, *Science Advances*, 6(21), p. eaaz8344. Available at: https://doi.org/10.1126/sciadv.aaz8344
2. Electrical Resistance as a measure of Growth
3. Meyer, A.J. *et al.* (2019) ‘Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors’, *Nature Chemical Biology*, 15(2), pp. 196–204. Available at: https://doi.org/10.1038/s41589-018-0168-3