The expected fluorescence(determined by the ratio of suboptimal structures with open/closed toehold domains) exhibited by E. coli co-transformed with the switch and trigger RNA after 12 hours was compared with the actual observed fluorescence during the experiment.
Experimental verification of the thermodynamic mode
Correlation of switch MFE (Minimum Free Energy) and expression leakage
The plot shows some kind of weak positive correlation between expression leakage and the MFE of a switch, with an outlier N9-2. This suggests that the MFE of a switch might not have a large contribution to the expression leakage.
Correlation of switch MFE (Minimum Free Energy) and the expression by Switch-Trigger duplex
Although the switch MFE correlates with the expression leakage, this plot showed that the switches with lower MFE also have lower RFP signal in the presence of trigger. This suggests that a low switch MFE could also be a hindrance to detection that it increases the energy input to the system (activation energy), so it takes more energy for switch-trigger duplex formation to occur. This could lower the expression level of the switch-trigger duplex.
Correlation of MFE difference and the expression by Switch-Trigger duplex
We initially thought that the larger the MFE difference between the sum of the MFE of switch and trigger and that of the switch-trigger duplex, the more favourable the duplex structure will be. However, there seems to be a weak positive correlation between the expression level of the switch-trigger duplex and the MFE difference of a switch. This could be because that a low MFE difference could indicate a more similar conformation of the switch/trigger RNA to the switch-trigger duplex RNA. This could lead to less conformational changes in the duplex formation process, making the process faster. Thus, a small MFE difference could lead to an increased expression level of the switch-trigger duplex.
Correlation of toehold domain base pair and the expression by Switch-Trigger duplex
The plot shows some kind of negative correlation between RFP signal in the presence of trigger and the number of base pairs in the toehold domain. Some exceptions were observed, they are N1-3, N9-3, N9-1. H5-3 and N1-2. This supports the assumption in the suboptimal structure modelling that an increasing number of unpaired bases in the toehold domain promotes initial binding of a trigger RNA to the corresponding switch RNA.
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