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 potassium ion, phosphate ion and nitrate (KPN) sensor, which aims to combine three constructs characterised by different outputs within one plasmid.

As the substitute of the KPN inducible promoters, 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 inducers. Two graphs of fluorescence changes according to different inducers concentrations for both single and double constructs could be analysed and compared.

Method

Molecular Cloning

There were two inducible single construct 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.

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

Experiment that we did

Characterization

As all of them were inducible constructs, fluorescence would only be expressed when inducers were present.

For every single characterization, same procedures were followed. First, the sample which would be measured had to be inoculated in Falcon tubes. In the next day, 25-fold dilution was carried out for the inoculated samples by using the M9 minimal medium with specific inducers and concentrations. They would then be transferred into 96-well deep well plate for overnight induction. Again, the sample would be further diluted by ten-fold dilution in the next day. Several hours were needed to let the cells grow from lag phase to log phase. The OD₆₀₀ should be around 0.4-0.7 ideally and kept constant for every trial. Ultimately, the result could be gathered with the help of EnVision® Multilabel Reader(OD₅₉₅) using filter 485/14nm FITC and 535/25nm FITC for excitation and emission measurement respectively. All data would be plotted as graphs for further analysis.

Preparing medium concentration

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

Result obtained

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-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, 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 decreased compared to the single construct induced araBADp-GFPmut3b system whether the coexpressed construc t was induced or not. We conjecture that it is possibly due to the limited cellular resources(eg. amino acids, enzymes required for gene translation/transcription) and the synthesized proteins (eg.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 charactered construct in a double construct, including that for the K, P and N construct. Thus further improvements should be made.

Further Improvements

Progress made in brief:

araBADp-GFPmut3b and lacZp-RFP characterisation
Measuring the RFU (GFPmut3b and RFP respectively) of the of E. coli samples, in increasing concentrations of respective inducers (for both L-arabinose and IPTG), using 3 biological replicates and 2 technical replicates.

araBADp GFPmut3b-lacZp RFP characterisation
Measuring the RFU (GFPmut3b and RFP respectively) of the of E. coli samples, in increasing concentrations of inducers, both L-arabinose and IPTG, using 3 biological replicates and 2 technical replicates.

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.