Expression platform

To examine the possible effects of co-expression, comparison between dose dependent fluorescence response expressed by one construct in a single plasmid and two constructs in a single plasmid was conducted . The purpose of the comparison relates to the design of potassium ion, phosphate ion and nitrate ion (KPN) sensor, which aims to combine three constructs characterised by different outputs within one plasmid.

The construction of a double construct araBADp-lacZp 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.

The construction of a double construct araBADp-GFPmut3b-lacZp-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.

Experiments performed

For all experiments, the sample to be measured was first inoculated in Falcon tubes. The next day, 25-fold dilution was carried out for the inoculated samples using M9 minimal medium with specific inducers and concentrations. They were then be transferred into a 96-well deep well plate for overnight induction. Again, the sample would be further diluted ten-fold the next day. The cells were allowed to grow from lag phase to log phase for several hours. The OD₆₀₀ was ideally around 0.4-0.7 and was kept constant for every trial. Ultimately, the result was gathered with the help of EnVision® Multilabel Reader(OD₅₉₅). All data were plotted as graphs for further analysis.

Please visit co-expression experiments for more details.

Results

After obtaining characterization data for the araBADp-GFPmut3b single construct, the dose response curve was compared with that expressed in a double construct (with lacZp-mRFP) .

From figure 5a, it can be observed that in a double construct, the maximum expression of the reporter decreases for the L-Arabinose inducible araBADp system. Furthermore, from figure 5b, it is shown that when the coexpressed system is under full induction, the curve for the double construct araBADp 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 coexpressed lacZp-mRFP construct on the GFPmut3b expression of the double construct araBADp system, experiments were carried out varying the IPTG induction concentrations.

Figure 6 exhibits that varying the induction level of the coexpressed lacZp-mRFP construct seems to have an effect on the expression level of araBADp-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, though at the highest IPTG concentration the graph observed to shift right, no significant shift was obtained at lower IPTG concentrations.

Conclusion

By using the araBADp-GFPmut3b and lacZp-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 decreased compared to the single construct induced araBADp-GFPmut3b, whether the coexpressed construct was induced or not. We conjecture that it is possibly due to the limited cellular resources(eg. aminoacids, enzymes required for gene translation/transcription) and the synthesised proteins (eg.lacI, mRFP) that could increase the cell load and affects its growth. However, since the investigation was done focusing only on one combination of inducible constructs(araBADp-GFPmut3b and lacZp-mRFP) it may not be used to generalise the effect of expressing a charactered construct in a double construct, including that for the K,P and N construct. Thus further improvements should be made.

Further Improvements

Improvements:

Since characterization data from one combination of double construct cannot be applied to all double constructs in general, characterisation of more combinations of inducible reporter systems (eg. luxRp-GFPmut3b- lacZp-mRFP, tetOp-GFPmut3b- lacZp-mRFP) is required.

Charaterize araBADp-GFPmut3b lacZp-RFP with a third construct (i.e. tetOp and luxRp)
This will give a more reliable comparison for a triple construct KPN promoter system, as opposed to a double system.

Characterize constructed potassium, phosphate and nitrate sensors with their respective inducers.