Part:BBa_K3143671

J23109-merR-pMerR-TEV-terminator

J23109-merR-pMerR-TEV-terminator is an Amplifier design for mercury sensor. In this design, Hg ions regulate the expression of TEV protease; the recognition site of TEV is inserted into C1434. When TEV protease presents, C1434 protein is cleaved by TEV and loses its activity. In the absence of TEV protease, C1434 inhibits the pR promoter and inhibits GFP expression at the transcriptional level. The final logic is that Hg ion concentration induces GFP expression. Due to the sensitivity of TEV itself, a small amount of expressed TEV protease can inactivate C1434, thereby allowing GFP expression. See more amplifiers design in EV-CI1434 BBa_K3143671 and Rinp80α-TEV-tevS-AAV Tag BBa_K3143669

Figure 1: A The scheme of Hg Amplifier using TEV-C1434 B Circuit design of Hg Amplifier using TEV-C1434

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.

• Amplifier1: Rinp80α

• Amplifier2: TEV-C1434

• Amplifier3: Rinp80α-TEV-tevS-AAV Tag

As is shown in Fig. 2, we can see：

• 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.

• 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.

• 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.



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 1: 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




Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 502
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 939
  Illegal SapI.rc site found at 1287