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 one plasmid was conducted . The purpose of the comparison relates to the design of nitrate, potassium ion and phosphate ion (NPK) sensor, which aims to combine three constructs characterised by different outputs within one plasmid.

As the substitute of the NPK 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

Cloning

There were two inducible single construct for characterization. They were araBADp with GFPmut3b and lacZp with mRFP induced by 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 5a, it can be observed that in a double construct, the maximum expression of GFPmut3b decreases for the 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 6 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.