Mini sub cell gt cell

Goat anti-mouse IgG (GTX77316, GeneTex), goat anti-rabbit IgG (GTX77061, GeneTex), streptavidin (85,878, Sigma Aldrich), streptavidin conjugation kit (ab102921, Abcam), Amicon® Ultra filters low-binding PES filter with 30 k molecular separation pores (UFC503096, Millipore), agarose powder (4,718, Sigma Aldrich), TAE buffer (Tris/Acetic Acid/EDTA) (1,610,743, Bio-Rad), SYBG Gold dye (41,003, Thermo Fisher Scientific), low–molecular weight DNA ladder (B7025, New England Biolabs Inc.), PowerPac™ basic power supply (1,645,050, Bio-Rad), Mini-Sub Cell GT Cell (1,704,406, Bio-Rad), Gel Doc™ EZ System (1708270EDU, Bio-Rad), SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices), recombinant human insulin (I2643, Sigma Aldrich), recombinant human IFN-γ (285-IF, R&D Systems), recombinant human IP10 (ab9810, Abcam), recombinant IL-6 (206-IL, R&D Systems), recombinant TNF-α (210-TA, R&D Systems), recombinant IGF-1 (291-G1, R&D Systems), recombinant human proinsulin C-peptide (NBP235211, Novus Biologicals), rabbit polyclonal anti-human insulin (ab53591, Abcam), rabbit polyclonal anti-human human insulin antibody (NBP1-87485, Novus Biologicals), rabbit polyclonal anti-human insulin (biotin) (ab53592, Abcam), mouse monoclonal anti-human insulin (ab6995, Abcam), and mouse monoclonal anti-human human insulin antibody (NBP100-73008, Novus Biologicals).

The yedQ (NCBI Gene ID: ID: 7149712, previously known as yhcK) gene from Escherichia coli IAI39 was cloned from the pYedQ plasmid (Ausmees et al., 2001; Chen et al., 2015) into the HindIII/BamHI side of pBBR1MCS‐5 plasmid vector to make the pYedQ₂ plasmid (Fazli et al., 2015). E. coli DH5α was transformed with the pYedQ₂ plasmid via heat shock (Hanahan et al., 1991). The S. oneidensis strain with an elevated c‐di‐GMP level (i.e. MR‐1/pYedQ₂) was constructed via plate mating (i.e. tri‐parental conjugation) using the following strains at mid‐logarithmic growth stage: S. oneidensis MR‐1 (recipient), E. coli HB101/pRK600 (helper) and E. coli DH5α/pYedQ₂ (donor). Further experimental details on plate‐mating can be found in Supplementary Information (Text S1). Agarose gel electrophoresis using Mini‐Sub^® Cell GT Cell (Bio‐Rad, Hercules, CA, USA) was run with the plasmid extracted from the selected mutant as a confirmation that the selected mutant indeed contains the pYedQ₂ plasmid. All swim plate experiments were done in 0.3% LB agar plates at 30°C for 24 h with five replicates for each sample (Fig. S1). CFU counts using LB agar plates with and without 50 µg ml⁻¹ gentamicin of MR‐1/pYedQ₂ culture grown for 12, 24 and 48 h showed insignificant differences (t‐test P‐value < 0.05, n = 3). No growth was observed for MR‐1 WT on LB agar plates with 50 µg ml⁻¹ gentamicin at 12, 24 and 48 h.

An 3% agarose gel electrophoresis was performed to evaluate the association efficiency of the miR. A known amount of miR (2 µg) was mixed with Loading buffer, Tris-Borate-EDTA (TBE) buffer and SYBR Gold. The agarose gel was prepared in TAE buffer, composed by Tris, acetic acid and EDTA 0.5 M. Prepared samples were loaded, and the gel was run at 80 V for 40 min, making use of a Mini-Sub Cell GT Cell (BioRad, California, United States). The result was evaluated with the ChemiDocTM MP Imaging System (Bio-Rad, California, United States), in which not-associated miR appears as a band in the gel. In the case of pDNA, 0.2 µg was loaded in a 1% agarose gel, following the same protocol.