Part:BBa_K3143668
PrinA_p80α-sfGFP-tevS-AAV Tag-terminator
PrinA_p80α-sfGFP-tevS-AAV Tag-terminator is the second part pf an advanced Amplifier design for mercury sensor. The first part is BBa_K3143669. This optimized design is still based on the RinA_p80α transcriptional activation system, but we also add the TEV-tevS-AAV Tag system to output GFP. In the system, TEV and RinA_p80α are regulated by Hg ions. At the same time, we fuse the GFP protein with the tevS-AAV tag. There are three factors control the expression of GFP. First, the activation of Ripn80α regulates GFP at the transcriptional level. Second, endogenous proteases of E. coli can recognize GFP-tevS-AAV Tag and degrade it. Third, TEV can recognize and cleavage tevS in GFP-tevS-AAV Tag. The site thus cuts off the AAV Tag site, and endogenous proteases no longer affect GFP expression.
Figure 1: Circuit design of Hg Amplifier 3 (RinA_p80α-TEV-tevS-AAV Tag)
As can be seen from Figure 2A & B, the yellow line represents the GFP-tevS-AAV Tag expression level, the blue line represents the expression of TEV, and the red line is the actual fluorescence output signal. When TEV is not expressed, it is degraded by endogenous proteases, which reduces the actual fluorescence output, alleviating the leakage of the Sensor; when TEV is gradually expressed, the GFP-tevS-AAV Tag is gradually cleavaged by TEV, making the fluorescence, or the output signal, gradually accumulate to higher level. Figure B shows Different TEV expression scenarios lead to different output.
Figure 2: A Different gene expression levels in Amplifier 3 (RinA_p80α-TEV-tevS-AAV Tag) B Different TEV expression scenarios lead to different output
Characterization
We introduced three amplifier designs between the sensor module and the reporter to improve the performance of mercury sensor. The relative design are described in Design.
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Amplifier1: RinA_p80α
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Amplifier2: TEV-C1434
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Amplifier3: RinA_p80α-TEV-tevS-AAV Tag
As is shown in Fig. 3, we can see：
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Amplifier1: Compared to Basic Sensor, the fluorescence output signal is much higher than Basic Sensor. However, he fluorescent signal leakage of this design at low mercury induction levels is very high.
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Amplifier2: Compared to Amplifier1, the fluorescent signal leakage of this design at low mercury induction levels is very low. However, the fluorescence output signal is much smaller than Amplifier1.
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Amplifier3: this design fully protected the GFP reporter from degradation at high mercury induction levels, while achieving significantly lower basal expression through continuous degradation of the reporter GFP at low mercury induction levels.
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 502
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1514
Illegal SapI.rc site found at 1862
Figure 3: Characterization of an mercury sensor with three different amplifiers
We fitted the sensors’ dose–response curves to a Hill function-based biochemical model to describe their input-output relationships (Table 2) . Here, both in Amplifier 1 & 2, EC50 showed a significant increase, while in Amplifier 3, EC50 deceased to the same level as Basic sensor (J23109). KTop showed a higher value in all three designs, in which Amplifier 1 & 3 are highest.
Table 1: Best fits for the characterized response of the various sensors circuits in this study
Figure 4: The maximum output (KTop) and EC50 of the sensor’s dose response with different amplifiers
The characterization of Amplifier 3 shows that this design fully protected the GFP reporter from degradation at high mercury induction levels, while achieving significantly lower basal expression through continuous degradation of the reporter GFP at low mercury induction levels.
In summary, Amplifier 3 is sufficient to reduce the sensor’s basal background while also being able to maintain both the sensor’s output amplitude and sensitivity, leading to expanded output dynamic range. What’s more, this strategy can also be applied to other heavy metal sensor circuits, such as As3+ (arsR)，Pb2+ (pbrR), etc.
Sequence and Features
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