Agarose

Cells (1 × 10⁴) were added to 2 ml growth medium with 0.5% agarose (Promega) and layered onto 2 ml 0.35% agarose‐supplemented medium in six‐well plates. The cells were fed with 1 ml growth medium weekly for 4–6 weeks, after which colonies were fixed with 10% acetic acid/10% methanol for 10 min, followed by staining with 0.005% crystal violet for 1 h, and counted using a light microscope. Colony numbers and sizes were determined using ImageJ v. 1.43 (NIH, USA).

Luria-Bertani (LB) broth for growing bacteria was purchased from Sigma-Aldrich (St. Louis, MO, USA). Agarose was from Promega (Madison, WI, USA). Silicon wafer was obtained from University Wafer (Boston, MA, USA). Agar, polystyrene petri dishes (diameter 100 mm), glass microscope slides, and coverslips were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Escherichia coli (E. coli) K-12 expressing enhanced green fluorescent protein (EGFP) was received as gift from Dr. Yongku Cho’s laboratory (University of Connecticut). The fluorescence from E. coli expressed EGFP was used only for validation purpose. Images were captured by Nikon A1R confocal microscope (Nikon, Japan) and processed using Nikon NIS-Elements Advanced Research microscope imaging software version 4.40.

The mtDNA control region (CD) was partially amplified by PCR using the following primers: R3 (L15926) THR 5′-TCA​AAG​CTT​ACA​CCA​GTC​TTG​TAA​ACC-3 (Kocher et al., 1989 (link)) and TDKD (H16498) 5′-CCT​GAA​GTA​GGA​ACC​AGA​TG-3’ (Slade et al., 1994). Amplifications for CR were carried out in 50 μl with one unit of Platinum DNA Taq polymerase (Invitrogen), PCR buffer 1X (Invitrogen), 2.0 mM MgCl₂, 0.25 mM dNTPs mix (Invitrogen), 0.5 μM of each primer, and 40 ng of template DNA. The thermocycling protocol consisted of an initial denaturation at 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 65°C for 60 s, 72°C for 60 s, and one final cycle of 72°C for 7 min. Amplification products were visualized in 2% agarose (Promega) gel electrophoresis using SYBR safe (Invitrogen) for staining. The amplicons were further purified with the UltraClean PCR clean up kit, according to the manufacturer’s instructions. The purified products were sequenced in both directions using an ABI 3730XLs sequencer (Macrogen, Inc., Seoul, South Korea). The sequences were edited using PreGap4 and Gap4 from the Staden software package (Staden et al., 2000 ) to obtain consensus sequences using information obtained from sense and antisense DNA strands. The sequences were then aligned using MEGA X (Kumar et al., 2018 (link)) with ClustalW algorithm and trimmed to obtain sequences of equal lengths.

For the soft agar assay, cells were trypsinized to generate single-cell suspensions and counted. 1.2% agarose (Promega, Madison, WI, USA) dissolved in medium was plated on the bottom of each well. Single-cell suspensions (1,000 cells per well) were mixed with 0.6% agarose and seeded on top of the bottom agar. All assays were performed in triplicate. The cells were incubated at 37°C for 6 weeks to allow colony formation. Images were captured using a Zeiss inverted wide-field microscope equipped with a Canon G12 camera.