Pureyield miniprep kit

The bacterial species were determined by 16S rDNA sequencing. Therefore, genomic DNA was isolated from 2 mL of liquid culture using the GenElute Bacterial Genomic DNA Kit (Sigma) and the 16S rDNA region was amplified by PCR using rD1 and fD1 primers (Weisburg et al., 1991 (link)). The resulting amplificate was cloned into pGEM-T vector (Promega, Madison, WI, United States), introduced into E. coli Alpha Silver Select Efficiency cells (Bioline, Luckenwalde, Germany). The plasmids were isolated using the PureYield Miniprep kit (Promega, Madison, WI, United States) and the insert was sequenced from both sides using T7 and SP6 primers (GATC, Konstanz, Germany). Vector fragments in the sequencing result were removed using VecScreen-Blast and the full 16S rDNA was assembled. BLASTn search of the 16S rDNA sequences revealed the closest relatives, thereby enabling determination of the species, due to the fact that identity to known type strains was in the range of 98–100%. The phylogenetic tree of all obtained bacterial isolates was created by aligning the 16S rDNA sequences of the respective type strains using MAFFT (Katoh et al., 2002 (link)) and constructing the tree using raxML (Stamatakis, 2014 (link)).

Flp-In 293 cells (catalog number R750-07), pCDNA5/FRT/TO, and pOG44 were obtained from Life Technologies. Flp-In 293 cells were maintained in DMEM high-glucose w/L-glutamine supplemented with an additional 2 mM L-glutamine (Gibco) and 100 µg/mL zeocin (Life Technologies). Haps59 and yCD were cloned into pCDNA5/FRT/TO, and DNA for transfections was prepped from overnight cultures of E. coli using PureYield Miniprep kit (Promega). Α total of 2.7 µg of pOG44 and 300 ng of pCDNA5/FRT/TO-EV, -yCD, and -Haps59 were transfected into 100,000 Flp-In 293 cells, plated eighteen hours prior in a single well of a 6-well plate using the CalPhos Mammalian Transfection Kit (Clontech) following the manufacturers directions. Twelve hours after transfection the cells were washed with PBS and fresh media was added. Thirty-six hours after transfection the cells were split 1 to 5 into a fresh 6-well plate of media containing 150 µg/mL hygromycin-B, instead of zeocin. Media was changed every 3–4 days until foci formed, in about 2 weeks. These stable pools were passaged 3 times, at a 1/10 dilution into 200 µg/mL hygromycin-B, before being used in toxicity assays.