Team:HKUST-Rice/Expression
Parallel Sensors
Expression platform
To examine the possible effects of co-expression, 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 (KPN) sensor, which aims to combine three constructs characterized by different outputs within a single plasmid.
Figure 1. Brief diagrams of single and double constructs.
The construction of a double construct ParaBAD-PlacZ allowed for a comparison between GFPmut3b and mRFP measurements while changing concentrations of the individual inducers. Graphs of changes in fluorescence according to different inducer concentrations for both single and double constructs could be analysed and compared.
Figure 2a. Schematic diagram of the inducible ParaBAD-GFP 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 GFP.
Figure 2b. Schematic diagram of the inducible PlacZ-mRFP construct. In the absence of Isopropyl b-D-1-thiogalactopyranoside (IPTG), LacI represses PlacZ. When IPTG is present, LacI protein is repressed, triggering the production of mRFP.
The construction of a double construct ParaBAD-GFPmut3b-PlacZ-mRFP allowed for a comparison between GFPmut3b and mRFP measurements while changing concentrations of inducers. Dose response curve for both single and double constructs could then be analysed and compared.
Constructs
Constructs were built by digestion and ligation method.
Figure 3. Inducible double construct ParaBAD-GFP-PlacZ-mRFP..
Figure 4. Inducible construct (a) ParaBAD-GFP and (b) PlacZ-mRFP..
Experiments performed
For all experiments, the bacteria were first grown overnight, then a 25-fold dilution was carried out using M9 minimal medium with specific inducers and concentrations. After that, they were transferred into a 96-well deep well plate for overnight induction. The samples were then further diluted ten-fold the next day. The cells were allowed to grow from lag phase to log phase for several hours. Ultimately, the result 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.
Results
After obtaining characterization data for the ParaBAD-GFPmut3b single construct, the dose response curve was compared with that expressed in a double construct (with PlacZ-mRFP).
Figure 5 . Response curve of ParaBAD-GFP in single construct compared with in double construct plasmid (a) Co-expressed construct (PlacZ-mRFP) with full induction (5mM IPTG) (b) co-expressed construct without induction.
From Figure 5a, it can be observed that in a double construct, the maximum expression of the reporter decreases for the L-Arabinose inducible ParaBAD system. Furthermore, from Figure 5b, it is shown that when the co-expressed system is under full induction, the curve for the double construct ParaBAD shifts to the right compared to both the single construct and double construct without induction, indicating a decrease in sensitivity.
In order to further investigate the effect of induction level of the co-expressed PlacZ-mRFP construct on the GFPmut3b expression of the double construct ParaBAD system, experiments were carried out using varying IPTG concentrations.
Figure 6 exhibits that varying the induction level of the co-expressed PlacZ-mRFP construct seems to have an effect on the expression level of ParaBAD-GFPmut3b in a double construct. At a higher IPTG concentration, the maximum expression of GFPmut3b decreases. However, no trend can be 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.
Conclusion
By using the ParaBAD-GFPmut3b and PlacZ-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 conjecture that it is possibly due to the limited cellular resources (e.g. amino acids, enzymes required for gene translation/transcription etc.) and the synthesized proteins (e.g. lacI, mRFP) that could increase the cell load and affect its growth. However, since the investigation was done focusing only on one combination of inducible constructs(ParaBAD-GFPmut3b and PlacZ-mRFP), the data obtained cannot be assumed to apply to all instances of expressing a charactered construct in a double construct, including that for the KPN construct. Thus, further improvements should be made.
Further Improvements
Since characterization data from one combination of double construct cannot be applied to all double constructs in general, characterization of more combinations of inducible reporter systems (eg. PluxR-GFPmut3-PlacZ-mRFP, PtetO-GFPmut3b-PlacZ-mRFP) is required.
Charaterize ParaBAD-GFPmut3b PlacZ-mRFP with a third construct (i.e. PtetO and PluxR)
This will give a more reliable comparison for a triple construct KPN promoter system, as
opposed to a double system.
Co-express potassium, phosphate and nitrate sensors and their respective inducers.
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.