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. This is an improvement part to BBa_K1758343.

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

NDNF_China 2021’s Characterisation

Heavy metal pollution in water bodies is a human-caused environmental issue that many governments and institutes tried to deal with. For example, Hg2+ is a well-known and widespread environmental contaminant that can adversely affect human health. To demonstrate that encapsulated bacteria can function in a real-world setting, we used an Hidro to detect the presence of metal ions in water samples. To learn more about the Hidro system, please visit <a href="https://2021.igem.org/Team:NDNF_China/Proof_Of_Concept">NDNF_China Proof-Of-Concept</a>.

We characterized the fluorescence output induced by Hg2+ in Hidro harbouring the genetic design BBa_ (Figure 1A). In the Hg2+ sensing circuits, fluorescent protein GFP can only be expressed when Hg2+ combines with the sensory protein, merR.

Hidro beads with bacteria harbouring the Hg2+ sensing circuits were incubated in liquid added Hg2+. As you can see from Figure b, exposure to 10μM Hg2+ resulted in the expression of GFP in E. coli characterized by plate reader after breaking beads and homogenization, indicating successful detection of Hg2+ ions. Thus, this result highlighted the potential of Hidro to detect toxic levels of heavy metals in Nature environment water bodies (Figure 1B).

(All experiments involving heavy metals follow the appropriate laboratory safety measures, please see more information in Safety)

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Figure 1: (A) The schematic of a Hg2+ sensing circuits in Hidro; (B) Hg2+ sensing circuits output.

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