Part:BBa_K3484004
AHL sfGFP+ASV reporter
This composite part produces sfGFP when a large enough concentration of lactone (AHL) is present in the medium. The construct 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.
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 and was characterized in E.Coli DH5α.
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
A model to characterize the AHL sfGFP+ASV reporter was designed. It must be considered that the sfGFP production follows a Hill function, as it is activated by a complex. Moreover, LuxR is constitutively expressed and due to the law of mass action it can be simplified (For more detail go to iGEM UPF_Barcelona 2020 Model section) . The final ODE system can be seen below (Eq.1).
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].
The transfer function (Eq.2) was fitted with experimental data to see if our model could follow real data. This data was extracted from a Plate-Reader analysis on GFP fluorescence emission with concentrations of lactone going from 100 pM to 100 µM. From these results we can conclude that there is a high correlation between the experimental and the modelling results, thus indicating that the AHL sfGFP+ASV reporter model we have developed reflects the real phenomena that happen in a sensor cell.
[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.
Characterization experiments
For the characterization a Plate-Reader analysis was made. All the information on the experimental conditions and parameters used are described on the table below (Table 1).
| Table 1. Plate-Reader Parameters for the characterization of the effects of ASV tag in sfGFP | |||
| Parameters | Value | ||
| Plate-Reader model | Synergy HTX | ||
| Plate type | Thermo Fischer 96-well microplates black-walled clear bottom | ||
| Cell medium | LB | ||
| Time | 24 hours | ||
| Shake | Linear: Continuous, Frequency: 567 rpm (3mm) | ||
| Temperature | 37C | ||
| Gain | 50 | ||
| Optical Density (OD) measurement (absorbance) | 660nm | ||
| GFP excitation wavelength | 485nm | ||
| GFP emission wavelength | 528nm | ||
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1249
Illegal AgeI site found at 1372 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1142
| None |