3.1. Strains and Plasmids

E. coli BL21 (DE3) and E. coli BL21 (DE3) pLysS were used for heterologous expression. E. coli DH5α was used for molecular cloning. The amino acid sequences of cGAS homologues and their corresponding nucleotide sequences were obtained from online databases (Table A1). The template pET-28 a (+) SUMOflhscGAS was cloned using the restriction sites NdeI and HindIII. From this template, the truncated human cGAS (thscGAS) and the SUMO sequence were amplified and fused with appropriate primers. The insert and vector pET-28 a (+) were cloned following the restriction ligation protocol with HindIII and NdeI as restriction sites. The gene sequences were synthesized by Eurofins and other homologues than hscGAS were cloned into pET-28 a (+) SUMOthscGAS using AflII and XhoI restriction sites or Gibson Assembly [36]. For construction of fusion proteins with superfolder green fluorescent protein (sfGFP), truncated SUMOcGAS genes were amplified with appropriate primers and cloned into pET GFP LICs cloning vector (linearized with EcoRV-HF) via Gibson Assembly or sequence and ligation-independent cloning (SLIC) [37]. The vector pET GFP LIC (u-msfGFP) was a gift from Scott Gradia (RRID:Addgene_29772; http://n2t.net/addgene:29772). The resulting plasmids were prepared using the NucleoSpin^® Plasmid (NoLid) Kit (Macherey-Nagel, Düren, Germany) for transformation of E. coli. For CFPS, plasmids were prepared using the GeneJET Plasmid Midiprep Kit (ThermoFisher Scientific, Waltham, MA, USA), followed by a second purification with the NucleoSpin^® Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany). A list of plasmids used in this study is shown in Table 1 and nucleotide sequences of the genes and primers are provided in Supplementary Materials 1.

Plasmids used in this study.