Part:BBa_K2581007
Inducible LuxR-pLux engineered device with weak RBS.
The UPF_CRG_Barcelona iGEM team 2018 has created this part as a intracellular biosensor of fatty acid uptake consisting in a high pass filter in order to reduce basal activity of the promoter. It consists of the assembly of a double terminator which allows forward and reverse terminator (BBa_B0014), a fatty acyl-CoA responsive promoter which expresses the downstream gene in the presence of fatty acids (BBa_k817002), a strong RBS based on Elowitz repressilator (BBa_B0034), luxR repressor/activator (BBa_C0062), the double terminator again (BBa_B0015), a weak RBS (BBa_B0032) and a reporter gene, an engineered mutant of red fluorescent protein from Discosoma striata (BBa_E1010).
Methods
E.coli bacteria (DH5-alpha) expressing the construct were induced with different concentrations of lux-homoserine lactone (HSL) and 3 different concentrations of PA (0, 0.4 and 1 mM) in LB medium. Fluorescence was analyzed once it had reached the steady state (11-13h). Fluorescence intensity values were normalized by the OD.
Characterization
In order to accurately measure LCFA uptake with the help of a genetic reporter, it is needed to obtain a significant signal rise after induction with LCFA. However, the fold change of the genetic expression triggered by the induction of the pfadBA promoter (BBa_K817002) is very low and does not allow a reliable quantification. We designed a system that allowed us to externally modulate genetic expression, which consists in the pfadBA promoter coupled to the LuxR/pluxR lactone (HSL) inducible system.
Our data indicates that the activation threshold of the HSL inducible pfadBA construct is approximately at 5x10-9 M of HSL. It saturates at 1x10-7 M of lactone. Significant fluorescence difference between different PA concentrations is better observed at lactone concentration ranges close to fluorescence saturation.
Fig. 1 | Fig. 2 | Comparison of fluorescence intensity between different pFadBA constructs. Bacteria were grown in LB media with different concentrations of PA (0, 0.4 and 1 mM). Bacteria expressing the pfadBA-plux construct were induced with lactone 1e-7 M. Fluorescence was analysed at steady state. Fluorescence intensity was normalized by the OD.
Conclusion
We achieved amplification of the output signal by inducing the pfadBA-luxR-plux-RFP with lactone 1e-7 M.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 102
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1764
Illegal AgeI site found at 1876 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1125
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