Part:BBa_K3143673

J23109-merR-pMerR-sfGFP-terminator

J23109-merR-pMerR-sfGFP-terminator is a basic design for mercury sensor. This sensorhas a constitutive promoter (J23109) that drives the expression of an mercury receptor MerR, which would de-repress its cognate promoter merR on murcury binding and trigger the expression of a reporter gene, GFP.

Figure 1: The scheme of basic sensor design.

Characterization

We select three constitutive promoters of varying strengths from iGEM promoter library (Fig. 2A). The sensors were then compared under various HgNO3 induction conditions (Fig. 2B). The results showed that the weaker the promoter (that is, the lower the MerR receptor concentration), the more sensitive and higher the dynamic range of the sensor.

Figure 2: A Different constitutively J23 family promoter measured strength (Data source: iGEM) B Tuning mercury receptor meRR’s intracellular density by varying the strength of J23 prmoter

We fitted the sensors’ dose–response curves to a Hill function-based biochemical model to describe their input-output relationships. (Fig 3a and Table 1)

• The Hill constant EC50 is the inducer concentration that provokes half-maximal activation of a sensor; EC50 is negatively correlated with sensitivity.

• KTop is the sensor’s maximum output expression level; KTop is positively correlated with output amplitude.

Figure 3: The equation used to fit the sensors’ dose–response curves to a Hill function based biochemical model to describe their input-GFPput relationships

Table 1: Best fits for the characterized response of the various sensors circuits in this study

Here, EC50 showed a 2.7-fold decrease and KTop showed a 3.5-fold increase from high to low MerR levels (Fig. 4A & 4B ), confirming that the mercury sensor’s sensitivity and output amplitude were both increased while the MerR intracellular concentration was decreased.

Figure 4: The maximum output (KTop) and EC50 of the sensor’s dose response against the relevant intracellular MerR concentrations

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 633