For the purpose of evaluating the optimal condition on testing the protein expression level with PURExpress in vitro protein synthesis kit, we calibrated the kit based on the intensity of sfGFP in BioBits™ Explorer, a commercially available toehold switch educational kit. To meet our particular needs, we conducted several experiments to optimize the ratio of solution A and B, which are the formula of the kit. Meanwhile, to lower the cost and to be more eco-friendly, we put efforts to lower down the total volume of our experiment. Initially, we tested conditions based on two references: the protocols of iGEM EPFL 2019 and the instruction manual in PURExpress protein synthesis kit.1
The ON/OFF ratio represents the quotient of the experimental group (with trigger RNA) and the control group (without trigger RNA). This ratio is how we evaluate the suitability of a toehold switch. For the first experiment (Condition A), we added the same ratio and amounts of reagents according to the instruction manual of PURExpress in vitro protein synthesis kit (Table 1). However, we found that the variance of the ON/OFF ratio at different time period was too low to be identified (Figure 1).
To tackle this problem, we referred to the experiment of former iGEM team, EPFL-2019, whose project also involved toehold switch and banana tohold sensor sfGFP. Therefore, taking their protocols into consideration, we altered the ratio between Solution A and Solution B to 0.75. Besides, awaring that the amount of total reaction volume suggested by the kit may cause leakage problem and over expression, we lowered it to one-fifth of the original one. In total, we tested four different conditions (Table 2) to figure out whether these changes could amplify the discrepancy of the ON/OFF ratio over time (Figure 2), and found the best situation based on this result.
According to our time-dependent experimental results, it is clear that the difference of ON/OFF ratio between the E condition and others was significant. Furthermore, for the production of reporter protein, the manual of in vitro protein synthesis kit suggests that the time duration is 120 mins. According to the result in figure 3, the ON/OFF ratio of the E condition at 120 mins was also the highest, which aligns with the recommendation from the kit and our need in the protocols for the ensuing experiments. (Figure 3)
In the E condition, we lowered the total reaction volume, raised the proportion of Solution B and DNA template. By modifying these three factors, we successfully tackled the leakage problem and made the variance of the ON/OFF ratio more significant. Therefore, the E condition of the in vitro protein synthesis kit is the optimal environment for our project.