Expression Platform

To examine the possible effects of parallel sensing, the dose-dependent fluorescence response of one construct in a single plasmid was compared to that of two constructs in a single plasmid. The purpose of the comparison relates to the design of our potassium, phosphate and nitrate (NPK) sensor, which aims to combine three constructs characterized by different outputs within a single plasmid.

As substitutes for the NPK-inducible promoters, P_araBAD and P_lac were used since their biological mechanisms are understood better.

The construction of a double construct P_araBAD - gfpmut3b - P_lac - mrfp allowed for a comparison between GFPmut3b and mRFP measurements while changing concentrations of inducers. Dose response curves for both single and double constructs could then be analyzed and compared.

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Constructs

Constructs were built by digestion and ligation method.

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Experiments Performed

For all experiments, the bacteria were first grown overnight and diluted 25-fold in M9 minimal media with specific inducers and concentrations. After that, the dilutions were transferred to a 96-well deep well plate for overnight induction. The samples were then diluted 10-fold the next day and were allowed to grow from lag phase to log phase for several hours. Ultimately, the measurements were gathered using a EnVision® Multilabel Reader. Data collected was plotted as graphs for further analysis.

Please visit Parallel Sensor Experiment Protocol for more details.

Experiments performed For all experiments, the bacteria were grown overnight and were diluted 25-fold in M9 minimal medium with appropriate inducer concentrations. After that, they were transferred into a 96-well deep well plate for overnight growth. The samples were further diluted ten-fold the next day and allowed to grow to log phase. Ultimately, fluorescence data was gathered using a EnVision® Multilabel Reader. Data collected was plotted as graphs for further analysis. Please visit Parallel Sensors Experiment Protocol for more details.

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Results

After obtaining characterization data for the P_araBAD - gfpmut3b single construct, the dose response curve was compared with the graph of the double construct (with P_lac - mrfp).

From Figure 5b, it can be observed that in a double construct, the maximum expression of the reporter decreases for the L-Arabinose inducible P_araBAD system. Furthermore, Figure 5c shows that the curve for the double construct P_araBAD shifts to the right when the co-expressed system is under full induction, indicating a decrease in sensitivity.

In order to investigate the effect of induction levels of P_lac - mrfp on the GFPmut3b expression in the double construct P_araBAD system, experiments were carried out using varying IPTG concentrations.

Figure 6 shows that variations in the induction level of the co-expressed P_lac - mrfp construct affects the expression level of P_araBAD - gfpmut3b in a double construct. At a higher IPTG concentration, the maximum expression of GFPmut3b decreases. However, no trend is observed for the horizontal shift of the curve except at the highest IPTG concentration (2² mM), where the graph shifts to the right. No significant shift was observed at lower IPTG concentrations.

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Conclusion

By using the P_araBAD - gfpmut3b and P_lac -mrfp inducible systems, the possible difference of dose-dependent fluorescence expression in a double construct and single construct was investigated. In a double construct, the maximal expression was lower than the single construct induced P_araBAD - gfpmut3b, whether or not the co-expressed construct was induced. We hypothesize that this is due to limited cellular resources (e.g. amino acids, enzymes required for gene translation/transcription etc.) since the synthesized proteins (e.g. lacI, mRFP) could increase the cell load and affect its growth. However, since the investigation focused on only one combination of inducible constructs (P_araBAD - gfpmut3b and P_lac - mrfp), the data obtained cannot be generalized to all instances related to expression of a characterized construct in a double construct, including those of the NPK constructs. Thus, further improvements should be made.

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Further Improvements

Since characterization data from one combination of double constructs cannot be applied to all double constructs in general, characterization of more combinations of inducible reporter systems (eg. P_luxR - gfpmut3b - P_lac - mrfp, P_tetO - gfpmut3b - P_lac - mrfp) is required.

Characterize P_BAD - gfpmut3b- P_lac - mrfp with a third construct (i.e. P_tetO and P_luxR) This will give a more reliable comparison for a triple construct NPK promoter system, as opposed to a double system.

Co-express potassium, phosphate and nitrate sensors and their respective inducers.

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References

Ang, J., Harris, E., Hussey, B. J., Kil, R., & McMillen, D. R. (2013). Tuning response curves for synthetic biology. ACS synthetic biology, 2(10), 547-567.

Wang, B., Barahona, M., & Buck, M. (2013). A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals. Biosensors and Bioelectronics, 40(1), 368-376.

Wang, B., Barahona, M., & Buck, M. (2015). Amplification of small molecule-inducible gene expression via tuning of intracellular receptor densities. Nucleic acids research, gku1388.