Contribution
We needed to induce IL-12 expression in response to lactate concentration in tissue microenvironment as high lactate levels is a hallmark of Warburg metabolism. For the same, we used the Part: BBa_K1847008 designed by iGEM15_ETHZ along with Strong, Medium and Weak RBS (BBa_B0032, BBa_B0034 and BBa_B0030 respectively) as we were interested in seeing the relative translation rates of our protein. Also, our part (BBa_K3110040) uses sfGFP (Part: BBa_I746916) instead of GFP as a reporter protein for characterization as it is reported to give more intense fluorescence and is more stable.
As our project requires our construct to discriminate against the higher lactate concentration in tumour micro-environment while remaining silent in normal tissue micro-environments, we planned to experiment with different combinations of the components, (Permease, Regulator and Dehydrogenase) of the lactate operon for modulating our system and attempted the constructs-lldD, lldP+lldD, lldR+lldD and lldP+lldR+lldD. We intend to use our mathematical modelling along with experimental data and literature to design a suitable O1-P-O2-RBS-sfGFP construct that is positive only in the cancer microenvironment. Our YebF-IL 12 sequence will then be inserted into the construct instead of sfGFP.
