pLux76B - dpB.013 |
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To complete our circuit, we need to engineer an output to our circuit that would allow us to measure the response of the processor. To connect our processor plasmid to our output plasmid we are using the marionette system [1], more specifically the inducer 3-oxohexanoyl-homoserinehlactone (OC6), that once produced by the processor would target the pLux76B promotor. To assess the strength and reliability of the connection between the two plasmid we needed to quantify the response of the promotor to its inducer. |
Materials and methods |
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We tested the impact of the inducer concentration on the level of gene expression to create a metric, allowing us to compare different output for when the system will be put in place. For that we transformed bacteria with a plasmid containing our inducer responsive promoter pLux76B coupled with a GFP. |
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We followed the evolution of the fluorescence over time using a Plate Reader. We set up in 100µL of bacteria per well in a 96-well plate, and performed serial dilutions from well to well to decrease the inducer concentration 10 fold per dilution, creating a range between 5 wells from 10M of OC6 in the first well to 10^-3 in the last. The bacteria were taken from an overnight culture and were at saturation. |
Results |
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When we plotted the evolution of the fluorescence over time per well (fig 1.), we could see clearly a difference in level of fluorescence for the different inducers concentrations. If we take the fluorescence values at around 3h, we can see that the bacterial fluorescence is proportional to the concertation of the inducers. (fig 2.) |
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When we plotted the evolution of the fluorescence over time per well (fig 1.), we could see clearly a difference in level of fluorescence for the different inducers concentrations. If we take the fluorescence values at around 3h, we can see that the bacterial fluorescence is proportional to the concertation of the inducers. (fig 2.) This reveals that the promoter is cleanly responsive to the inducer, proving that connecting the processor and the output using Marionettes [1] is a reliable way to pass on the signal. |
Figure1: Evolution of pLUXB induced fluorescence for varied OC6 concentrations over time
Figure2: Induced fluorescence in response to inducer concentration after (3h)
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