Escherichia coli stellar

The mitochondrial-targeting sequence 4mt, encoding four copies of the signal sequence from subunit VIII of human cytochrome C oxidase, was amplified using the Advantage Polymerase (Clontech, Mountain View, CA, USA) and primers F, 5′-ACTATAGGGAGACCCAAGCTTATG-3′ and R, 5′-TGGTGGCGGCAAGCTTCTTGCTCACCATGGTGGC-3′. The pcDNA-4mt-D3-cpv vector used as a matrix for amplification of 4mt was a kind gift from Dr. Roger Tsien (HHMI investigator at the University of California San Diego, San Diego, CA, USA). The PCR fragment was cloned into the HindIII restriction site of pcDNA3-Epac-S^H187 using the Infusion HD Cloning System (Clontech). Epac-S^H187 encodes for a fourth-generation Epac1-based cAMP sensor and was a kind gift from Dr. Kees Jalink (The Netherlands Cancer Institute, Amsterdam, Netherlands).^{36 (link)} Once the pcDNA-4mt-Epac-S^H187 vector was amplified in Stellar Escherichia coli (Clontech) bacteria, its identity with parental sequences was verified by PCR using primers F, 5′-ACTCACTATAGGGAGACC-3′ and R, 5′-TGCGGCCGCCATGGTGGC-3′, and DNA double-strand sequencing (INSERM U1056 – UMR 5165 CNRS UPS – UDEAR, Toulouse, France). Adenoviruses encoding Epac-S^H187 and 4mt-Epac-S^H187 were generated by Welgen Inc (Worcester, MA, USA).

FAM20C-pcDNA3.1/myc-His(-)A was generated from human cDNA encoding residues 1–584 of FAM20C (clone ID: 4942737, GE Healthcare). FAM20C cDNA was amplified with the following primers; forward: 5′-ACCCAAGCTGGCTAGATATGAAGATGATGCTGGTGCGCCG-3′ and reverse: 5′-GTTCGGGCCCAAGCTTCCTCGCCGAGGCGGCTCTG-3′ containing a NheI and a HindIII restriction sites, respectively (LGC Biosearch Technologies). Amplified cDNA was recombined into pcDNA3.1/myc-His(-)A (Invitrogen) using the InFusion Cloning Technology (Clontech). Plasmid was propagated in Stellar Escherichia coli (Clontech) followed by purification with the PureLink HiPure Plasmid Filter Maxiprep (Invitrogen). MEF3T3-275-3-2 cells were transfected at approximately 70% confluence where the medium was changed to serum-free medium in T25 culture flasks. Four micrograms of FAM20C-pcDNA3.1/myc-His(-)A was reacted with Lipofectamine LTX and Plus reagent (Invitrogen) and supplied to the cells. Conditioned medium was harvested after 48 h.

M. smegmatis wild-type strain mc²155 and its derivatives were grown in tryptic soy broth + 0.05% Tween 80 (TSBT) or on TSA plates, and cultured at 37°C. Antibiotic selection for reporter maintenance or mutation selection strategies included apramycin (12.5 μg/ml on agar, 10 μg/ml in broth), hygromycin (100 μg/ml and 25 μg/ml, respectively), kanamycin (50 μg/ml and 10 μg/ml, respectively), and zeocin (50 μg/ml and 25 μg/ml, respectively). Escherichia coli Stellar (Clontech) and NEB5-alpha (New England BioLabs) cells were used for transformation for all plasmid constructions using the Inoue method (55 (link)).