Expression platform

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

To substitute the KPN inducible promoters, two well-characterised promoters, araBADp and lacZp were used, as their biological mechanisms are better understood.

The construction of a double construct araBADp-lacZp allowed for a comparison between GFPmut3b and RFP 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.

Methods

Molecular Cloning

There were two inducible single constructs for characterization. They were araBADp with GFPmut3b and lacZp with mRFP, induced by L-arabinose and IPTG respectively. Both of the constructs were built by digestion and ligation method following the RFC10 standard.

The same approach was also adopted for the double construct to ligate araBADp and lacZp together.

Experiments performed

Characterization

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) using 485/14nm FITC and 535/25nm FITC filters for excitation and emission measurement respectively. All data were plotted as graphs for further analysis.

Preparing medium concentration

M9 minimal medium was used for inoculation, induction and dilution. It was chosen because of its low auto-fluorescence. Serial dilution was usually adopted for making medium with different concentration of inducers.

Results

Characterization data for the araBADp-GFPmut3b single construct, the dose response curve was compared with the same construct coexpressed in a double construct (with lacZp-RFP).

From Figure 4a, it can be observed that in a double construct, the maximum expression of GFPmut3b 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.

In order to further investigate the effect of induction level of the coexpressed lacZp-RFP construct on the GFPmut3b expression of the double construct araBADp system, experiments were carried out varying the IPTG induction concentrations.

Figure 5 exhibit that varying the induction level of the coexpressed lacZp-RFP 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 level 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, there is no significant shift at lower IPTG concentrations.

Conclusion

By using the araBADp-GFPmut3b and lacZp-RFP 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 always lower compared to the single construct induced araBADp-GFPmut3b system whether the coexpressed construct was induced or not. We conjecture that it is possibly due to the limited cellular resources (e.g. amino acids, enzymes required for gene translation/transcription) and the synthesized proteins (e.g. LacI, RFP) that could increase the cell load and affects its growth. However, since the investigation was done only focusing on one combination of inducible constructs(araBADp-GFPmut3b and lacZp-RFP) it may not be used to generalize the effect of expressing a characterized construct in a double construct, including that for the K, P and N construct. Thus further improvements should be made.

Further Improvements

Improvements:

Repeat number of experiments and biological and technical replicates until data is reliable and accurate.
With reliable and accurate data, conclusion made from this experiment can be used for further investigation.

Increase number of concentrations of inducers used within the same range to obtain detailed data from our experiments.

Characterize PLux GFPmut3b and PLux GFPmut3b- lacZp RFP.
Measuring the RFU (GFPmut3b and RFP respectively) of the of E. coli samples, in increasing concentrations of inducers, HSL and IPTG respectively, and HSL and IPTG respectively.

Summary of two promoter system in one construct we want to work on:

Charaterize araBADp-GFPmut3b lacZp-RFP with a third promoter system (i.e. PTet and PLux)
This will give a more reliable comparison of more than one promoter system, as opposed to one system.

Change order of the three promoter system and characterize the each construct, by measuring their RFU in increasing concentration of the three inducers L-arabinose, IPTG and ATC)
After comparing results of the three types of promoter system within each other, we can apply the data to the projects’ actual sensors and make a hypothesis of if combining the three sensors will affect the actual results.

Furthermore, we can start on characterizing the threes sensors in one promoter construct, 2 promoter construct, and three promoter construct and compare their results. We can do this by using similar methods as used above.

Compare two promoter system as opposed to one, using the Nitrogen, Phosphate, and Potassium sensors by characterizing them with their respective inducers.

Compare three promoter system as opposed to two and one using the Nitrogen, Phosphate, and Potassium sensors by characterizing them with their respective inducers.

Analyze data from all the characterization and come to a further conclusion that if one promoter system is significantly different from two promoter system or three promoter system.