Part:BBa_K3143672
J23109-merR-pMerR-RinA_p80α-terminator
J23109-merR-pMerR-RinA_p80α-terminator is an Amplifier design for mercury sensor. In this design, Hg ions directly control the expression of RinA_p80α activator, and the activation promoter Prina_p80α of RinA_p80α activator controls the output GFP. When Hg ions activate the expression of RinA_p80α activator, RinA_p80α activator will activate GFP expression on high-copy promoters, leading to a higher level of fluorescence values. See more amplifiers design in EV-CI1434 BBa_K3143671 and RinA_p80α-TEV-tevS-AAV Tag BBa_K3143669
Figure 1: Circuit design of Hg Amplifier using RinA_p80α
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. 2, 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 518
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 977
Illegal SapI.rc site found at 1325
Figure 2: 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 2: Best fits for the characterized response of the various sensors circuits in this study
Figure 3: 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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