Engineering Red Ca2+ Sensor: Pink Flamindo

The DNA fragment of a red fluorescent protein variant, mApple, utilized in a red Ca²⁺ indicator R-GECO^{12 (link)} was created by DNA synthesis from Integrated DNA Technologies (Coralville, IA, USA). It was engineered to contain SacII and EcoRI restriction enzyme sites between A150 and V151 and cloned into the pRSET-A vector. The cAMP binding domain of mEpac1 (NM_001171281, 199–358 amino acids) derived from Flamindo2 and was inserted into the SacII/EcoRI sites of mApple. To improve the performance of the indicator, a number of variants were created with the addition or deletion of linker amino acids in the both N-terminus and C-terminus of the cAMP binding domain by PCR. Random, site-directed mutations were also introduced into the construct by PCR with sense and anti-sense primers including NNK and MNN, respectively. The mutant construct that elicited the highest dynamic range was selected and named Pink Flamindo. Pink Flamindo was cloned into the pcDNA3.1 (-) vector for expression in mammalian cells. The humanised photoactivated adenylyl cyclase from Beggiatoa sp. (h_bPAC) in a pGEM-HE vector was obtained from Addgene (#28134, Cambridge, MA, USA), and was subcloned into the BglII/EcoRI sites of the pEGFP-C1 vector. The G-GECO plasmid was also obtained from Addgene (#32447). The Flamindo2 plasmid was developed in our previous study^{9 (link)}.