Dh5alpha cells

Wild type (WT) zebrafish TRPM7 (NP_001025232) was a kind gift from Dr. Michael Elizondo. WT drTRPM7 was restriction digested NotI/NheI and cloned into pcDNA5/TO (Invitrogen) with a hemagglutinin (HA) epitope tag at the N-terminus. All the subsequent mutant versions were cloned into HA-tagged pcDNA5/TO (Invitrogen) for tetracycline/doxycycline-regulated expression using either NotI/SpeI or NotI/KpnI restriction sites. Post cloning, sequencing was carried out to ensure that the coding sequence was in-frame for each construct.
To amplify cDNA for transfection of tetracycline-inducible HEK293 T-REx cells, competent DH5 alpha cells (Invitrogen Cat. No. 18258-012) were transformed with a pcDNA5/TO plasmid (Invitrogen Cat. No. V1033-20) harboring either drTRPM7 wild type (drTRPM7 WT), drTRPM7-1478, drTRPM7-1258 or drTRPM7-1178 truncations.

Midgut microbiota families were identified 24 h post-blood meal in each of the three aforementioned mosquito species that were reared in a mixed culture. After dissection, midguts were transferred into separately labelled tubes and kept on ice until the species of mosquito was identified using the PCR method described above. Ten midguts of the same species were pooled, and genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega, Madison, USA), according to manufacturer’s instructions. Purified gDNA was resuspended in 50 μl of sterile water before PCR amplification. 16S rRNA gene specific primers, 1492r and 27f as described in [32 (link)] were used to amplify partial sequences (>750 bp) of a 16S ribosomal gene from bacterial isolates. Products were cloned into the PCR-Blunt Vector and used to transform DH5-alpha cells (Invitrogen). For each mosquito species, 100 white colonies were selected randomly, and the 16S PCR product was sequenced. ClustalW and NCBI blast software was used to identify the bacterial family from the 16S rRNA sequences.

GST-RBF1-large pocket (LP), GST-RBF1-small pocket (SP), and GST-RBF1-C-terminus (C-term) constructs were generated using the pGEX-5x-1 vector (GE Healthcare Life Sciences) backbone digested with BamH1/Xho1 (NEB). LP, SP, and C-term segments of RBF1 was generated by using cDNA of full length RBF1. BamH1 and Xho1 sites were added to primer ends for successful ligation into vector. Primer sequences used for cloning is the following:
Successfully ligated vectors with appropriate inserts were then transformed into BL21 cells (NEB). Empty pGEX-5x-1 vector was used as a GST-alone control which was transformed into DH5-alpha cells (Thermo Fisher).