Team:CSMU Taiwan/Measurement

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Measurement






Introduction

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

Result

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).

Total Reaction Volume (μL) Volume Ratio - Solution B / Solution A DNA Template Amount (ng) Solution A Volume (μL) Solution B Volume (μL)
Condition A
(with trigger RNA)
25 0.4 250 12.5 5
Condition A
(without trigger RNA)
25 0.4 0 12.5 5
Table 1. Details for Condition A

Note: Solution A contains tRNA and small components such as amino acids and rNTP. Solution B contains ribosomes and protein components such as T7 RNA polymerase, amino-acyl synthetase, or energy regeneration enzyme.


Figure 1. The time-dependent analysis of the ON/OFF ratio in condition A.


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.

Total Reaction Volume (μL) Volume Ratio - Solution B / Solution A DNA Template Amount (ng) Solution A Volume (μL) Solution B Volume (μL)
Condition B
(with trigger RNA)
25 0.75 250 10 7.5
Condition B
(without trigger RNA)
25 0.75 0 0 7.5
Condition C
(with trigger RNA)
15 0.75 150 2 1.5
Condition C
(without trigger RNA)
15 0.75 0 2 1.5
Condition D
(with trigger RNA)
5 0.75 30 2 1.5
Condition D
(without trigger RNA)
5 0.75 0 2 1.5
Condition E
(with trigger RNA)
5 0.75 50 2 1.5
Condition E
(without trigger RNA)
5 0.75 0 2 1.5
Table 2. Details for different conditions.


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Figure 2. The time-dependent analysis of ON/OFF ratio in different conditions.


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)

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Figure 3. The bar chart for the ON/OFF ratio at 0, 60, and 120 mins in different conditions.

Conclusion

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.

Numeric Number Representations
Total Reaction Volume (μL) 5 By lowering the total reaction volume from 25 μL to 5 μL, we can conduct our experiment in an eco-friendly consideration.
Volume Ratio - Solution B / Solution A 0.75 By increasing the proportion of Solution B, the ON/OFF ratio can be increased over time.
DNA Template Amount (ng) 50 The toehold switch expression system can only function under an appropriate amount of DNA template.
Solution A (μL) 2 Downsized from its original 25 μL reaction, sufficient amount of Solution A is important in maintaining the expression level of reporter protein.
Solution B (μL) 1.5 Downsized from its original 25 μL reaction, sufficient amount of Solution B is important in maintaining the expression level of reporter protein.
Table 3. Summary of the Adjustments in Condition E





Reference


  1. Tuckey, C., Asahara, H., Zhou, Y., & Chong, S. (2014). Protein synthesis using a reconstituted cell-free system. Current protocols in molecular biology, 108, 16.31.1–16.31.22. https://doi.org/10.1002/0471142727.mb1631s108