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.
Figure 1. Brief diagrams of single and double constructs.
As substitutes for the NPK-inducible promoters, ParaBAD and Plac were used since their biological mechanisms are understood better.
Figure 2a. Schematic diagram of the inducible ParaBAD - gfpmut3b construct. In the absence of L-Arabinose, AraC is constitutively produced, repressing ParaBAD. In the presence of L-Arabinose, AraC protein driven by the constitutive promoter binds with L-Arabinose and induces ParaBAD, triggering the production of GFPmut3b.
Figure 2b. Schematic diagram of the inducible Plac - mrfp construct. In the absence of Isopropyl b-D-1-thiogalactopyranoside (IPTG), LacI represses Plac. When IPTG is present, LacI protein is repressed, triggering the production of mRFP.
The construction of a double construct ParaBAD - gfpmut3b - Plac - 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.
Constructs were built by digestion and ligation method.
Figure 3a. Inducible single constructs ParaBAD - gfpmut3b.
Figure 3b. Inducible single constructs Plac- mrfp.
Figure 4. Inducible double construct ParaBAD - gfpmut3b - Plac - mrfp.
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.
After obtaining characterization data for the ParaBAD - gfpmut3b single construct, the dose response curve was compared with the graph of the double construct (with Plac - mrfp).
Figure 5 . Response curve of ParaBAD - gfpmut3b in single construct compared to the double construct plasmid (a) Constructs used in the experiment (b) Co-expressed construct (Plac - mrfp) with full induction (5 mM IPTG) (c) co-expressed construct without induction.
From Figure 5b, it can be observed that in a double construct, the maximum expression of the reporter decreases for the L-Arabinose inducible ParaBAD system. Furthermore, Figure 5c shows that the curve for the double construct ParaBAD 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 Plac - mrfp on the GFPmut3b expression in the double construct ParaBAD system, experiments were carried out using varying IPTG concentrations.
Figure 6 shows that variations in the induction level of the co-expressed Plac - mrfp construct affects the expression level of ParaBAD - 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.
By using the ParaBAD - gfpmut3b and Plac -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 ParaBAD - 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 (ParaBAD - gfpmut3b and Plac - 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.
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. PluxR - gfpmut3b - Plac - mrfp, PtetO - gfpmut3b - Plac - mrfp) is required.
Characterize PBAD - gfpmut3b- Plac - mrfp with a third construct (i.e. PtetO and PluxR) 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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