Team:NUS Singapore-Sci/Improve

Improvement of Parts

The EGFP-T2A-mCherry bicistronic reporter is a significant improvement over the reporter system from a previous iGEM team.
An important feature of a reliable reporter system is to have an internal control signal such that the reporter signal can be normalized to account for stochastic processes in cells. Expression of both EGFP and mCherry reporter on separate plasmids may lead to uneven gene expression due to stochastic processes. Thus, it is more advantageous to have both EGFP and mCherry under the control of same promoter. Therefore, we designed our reporter system with this intention in mind. We then carried out experiments to compare our reporter system with the reporter system described by the Worcester 2016 team. We evaluated the system in terms of background noise, as well as the reliability of the internal control.

We performed double transfection of the mCherry plasmid with either ATG EGFP (BBa_K2083009) or ACG EGFP (BBa_K2083010) plasmid in HEK293T cells. As shown in Figure 1, WPI reporters showed the expected OFF to ON change from ACG mutant to ATG. However, the fluorescence intensity of EGFP and mCherry does not correlate well with one another, and there were some leaky expression of EGFP even in the mutant form (Figure 1C). Moreover, 2.3% of the cells are double positive for EGFP in cells expressing the WPI ACG mutant reporter (Figure 2B), which is higher than our ACG bicistronic reporter construct (as shown in Figure 3B in Demonstration).

On the other hand, our bicistronic reporter construct showed a strong linear correlation between EGFP and mCherry fluorescence intensity in WT reporter (as shown Figure 3A in Demonstration), and the number of cells positive for EGFP in our ACG mutant construct is effectively non-detectable. Therefore, our dual reporter system provides higher signal to noise ratio and allows for the quantification of relative editing efficiency between different cells and in different transfection experiments.



Figure 1. Fluorescence imaging of WPI 2016 EGPF reporters in transfected cells. HEK293T cells in a 6-well plate are transfected with 1ug of EGFP and mCherry plasmids each, and photos were taken 24 hrs post transfection. (A) EGFP expression in wild type ATG EGFP transfected cells. (B) mCherry expression when co-transfected with EGFP. (C) EGFP expression in mutant ACG EGFP transfected cells. There are a few cells with weak EGFP expression. (D) mCherry expression when co-transfected with EGFP. Images taken at 40X magnification, scale bar = 100 um.


Figure 2. Flow cytometry analysis on EGFP and mCherry expression on Worcester 2016’s wild type and ACG mutant reporters. HEK293T cells were co-transfected with wild type or mutant EGFP. (A) No clear correlation was observed between expression level of EGFP and mCherry. (B) Leaky expression of EGFP is observed in mutant ACG reporter. (C) Percentage of EGFP mCherry double positive cells. (D): Quantification on mean fluorescent intensity. Error bar represents SEM, n=3.
Further results using the Dual-Color Reporter system can be found in our Demonstration page.