Part:BBa_K3440021

MtrB under oscillatory circuit

Plux(BBa_R0062)-RBS(BBa_B0034)-MtrB(BBa_K3146001)-DT(BBa_B0015)-Pconst(BBa_J23100)-RBS(BBa_B0034)-RhlR(BBa_C0071)-DT(BBa_B0015)-Prhl(BBa_R0071)-RBS(BBa_B0034)-RhlI(BBa_C0070)-RBS(BBa_B0034)-AiiA(BBa_C0060)-RBS(BBa_B0034)-LuxR(BBa_C0062)-DT(BBa_B0015)

Usage and Biology

This is the final construct of our modular biosensor that includes an E.coli module producing a quorum sensing molecule upon detection of a pollutant and a Shewanella oneidensis mtrb knockout module that receives the quorum sensing molecule, triggering an oscillatory circuit under which electricity production is placed.

This part is the oscillatory circuit to be placed and induced in Shewanella Oneidensis MR-1 mtrb knockout.

Due to the leakiness of Prhl, RhlI is expressed and produces C4-HSL. Parallely, RhlR is constitutively expressed. The complex RhlR:C4-HSL induces Prhl. AiiA under Prhl degrades C4-HSL. Thus, with no AHL input, the circuit oscillates.

With a 3OC6-HSL input (produced by LuxI and used as quorum sensing molecule), this part can be used to produce electricity in an oscillatory manner thanks to expression of Mtrb. Indeed, LuxR is also expressed under Prhl and the complex 3OC6-HSL:LuxR activates Plux, the promoter under which Mtrb is placed.

Theoretical expectations

The output of this part was modeled by a system of delayed differential equations (detailed in [the model page of our wiki] (https://2020.igem.org/Team:Stockholm/Model). The results obtained below show that the oscillations of the system are active from the beginning, but no significant level of MtrB (“X”) is produced. This allows the cells to synchronize as the levels of C4-HSL (“H”) is oscillating from the very start, regardless of 3OC6-HSL (“H2”) signals. When 3OC6-HSL is added to the system, the levels of MtrB oscillates with the rest of the system at much higher levels.

Improved inducible oscillatory circuit at different induction points of 3OC6-HSL. Concentrations of RhlI (I), AiiA (A), MtrB (X), C4-HSL (H), 3OC6-HSL (H2) and LuxR (R) plotted over time

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 2324
  Illegal NheI site found at 2347
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4050
  Illegal BamHI site found at 2618
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 4181
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3093