Composite
AHL sfGFP

Part:BBa_K3484004

Designed by: Tomas Berjaga, Quim Marti & Jaume Puig   Group: iGEM20_UPF_Barcelona   (2020-10-26)
Revision as of 12:07, 26 October 2020 by Tomasberjaga (Talk | contribs)


AHL sfGFP+ASV reporter

This composite part produces sfGFP when a large enough concentration of lactone (AHL) is present in the medium. In this case, it constitutively produces LuxR, which binds to the lactone present in the medium and together, they form a complex that activates the pLux promoter, generating the sfGFP whose fluorescence can be measured. The schematics of this composite part can be seen in Figure 1.

Figure 1. Scheme of the interactions on the AHL sfGFP+ASV tag reporter sensor cell.

This system allows a fast report on the AHL concentrations in the medium, something important to know the amount of protein. The composite part is Biobrick compatible.

This composite part is composed by all the following parts:

BBa_B0015: A reliable double terminator that consists on BBa_B0010 and BBa_B0012.

BBa_B0033: A weak RBS. The RBS.4 (derivative of BBa_0030).

BBa_B0032: A medium RBS. The RBS.3 (medium) (derivative of BBa_0030

BBa_R0062: LuxR & HSL regulated promoter.

BBa_J72005: Ptet Promoter. Is activated by the AHL-LuxR complex

BBa_K3484006: sfGFP+ASV coding sequence


Model Characterization

From the ODE system (Eq.1) a transfer function can be derived if LuxR and GFP are on a steady state (dy/dx=0). This transfer function (Eq.2) allows us to connect the concentration of lactone (AHL) in the media to the fluorescence that the sensor cells are emitting. The function has the shape of a Hill function with a low hill coefficient (n=1), thus meaning that the sensor will not be hypersensitive and it will have a long dynamic range where it can sense the AHL concentration[1][2].

Equation 2. Transfer function of the AHL sfGFP+ASV biosensor model

[1] Ballestero, M. C., Duran-Nebreda, S., Monta, R., Solé, R., Macía, J., Rodríguez-Caso, C. A bottom-up characterization of transfer functions for synthetic biology designs: lessons from enzymology. Nucleic Acids Research, 2014, Vol. 42, No. 22.

[2] Garcia-Ojalvo, J., Elowitz, M.B., Strogatz, S.H. Modeling a synthetic multicellular clock: repressilators coupled by quorum sensing. Proc. Natl Acad. Sci. U.S.A., 101, 10955–10960.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1249
    Illegal AgeI site found at 1372
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1142


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Categories
Parameters
None