Part:BBa_K2581011
Improved fatty acid acyl-CoA biosensor with weak RBS
The UPF_CRG_Barcelona iGEM team 2018 has create this part as an improved element from the existing fatty acid intracellular promoter pfadBA (BBa_k817002). It consists of an intracellular biosensor of fatty acid uptake with a reduced basal activity that the mentioned promoter has. This biobrick consists in the assembly of a double terminator which allows forward and reverse terminator (BBa_B0014), our improved promoter based on the previous pfadBA DNA sequence (BBa_K2581013), 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 T14_pAR+32+RFP and T14_pFadBA_34_RFP constructs were induced with different concentrations of PA (0mM, 0.4mM and 4mM in LB media. Fluorescence was analyzed once it had reached the steady state(13-15h) and normalized by OD600.
Characterization
pFadBA (BBa_K817002) is a long chain fatty acid (LCFA) inducible promoter with excessive leakage and a poor dynamic range. Thus, this does not allow to see a significant rise in the signal after induction with LCFA. Here, we have characterized an improved version of the same promoter.
After pAR induction with different PA concentrations, our results show a significant fluorescence fold change increase compared to the pfadBA promotor. Also, our data indicates that the baseline fluorescence of the pAR construct is much lower than the pfadBA (Fig. 1 and 2).
Fig 1 | E. coli bacteria (DH5-alpha) expressing the construct were induced with different concentrations of PA in LB media. Fluorescence was analyzed once it had reached the steady state (13-15h). Fluorescence intensity values were normalised by the OD.
Fig 2 | Comparison of fluorescence intensity fold change (induced/non induced) differences between pFadBA and pAR inducible constructs.
Conclusion
pAR promoter significantly improves pfadBA function as an off/on switch dependent on the presence of LCFA
Our results show that the pAR promoter has a sharper response after PA induction compared to the pfadBA promoter. Moreover, our data suggests that it saturates at much lower PA concentrations than the pfadBA promoter and it has less leakage. Taking all this together, we can affirm that our newly characterised promoter would work much better as an off/on switch dependent on LCFA than the pfadBA promoter, therefore we have successfully improved a previous iGEM parts registry part.
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 761
Illegal AgeI site found at 873 - 1000COMPATIBLE WITH RFC[1000]
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