Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Microfluidic Evolution-On-A-Chip Reveals New Mutations that Cause Antibiotic Resistance.

doi: 10.1002/smll.202007166

Figure Lengend Snippet: Figure 5. Composition of E. coli cultures in the chip. E. coli cells were cultivated in chips for 8 days. a) The mature culture inside the chip com- prised a biofilm-like (BFL) material (I) which was flushed out using air, collected and quantified using crystal violet (CV) staining. A biofilm (BF) biomass remained attached to the chip surface (II) and could also be stained by CV. The BF was subsequently removed using 33% v/v acetic acid leaving the chip empty (III). b) The total BF and BFL biomass were directly compared using the colorimetric CV assay, which correlates bio- mass to the absorption at 585 nm. Inset: The BFL material removed from the chip in (I) is intensely opaque and viscous. For a microscopic analysis see Figure S6, Supporting Information. Error bars are the standard devia- tion (SD) of four samples derived from two independent chip replicas.

Article Snippet: ALE on Chip: The lab model bacterium E. coli K12 sub-strain MG1655 DSM 18 039 (DSMZ GmbH, Germany) harboring a kanamycin-selection plasmid (pMK-based backbone, lab stock) was used for all biological experiments.

Techniques: Staining, Derivative Assay

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Microfluidic Evolution-On-A-Chip Reveals New Mutations that Cause Antibiotic Resistance.

doi: 10.1002/smll.202007166

Figure Lengend Snippet: Figure 4. Influence of the flow profile on biomass growth pattern. a) Upon inoculation, the bacterial growth is always initiated in the wells (marked yellow), not in the channels. b) This corresponds well with the simulation of velocity profiles inside the chip using the COMSOL Multiphysics software, demonstrating non-homogeneous velocities in the wells, with high and low velocities indicated by white and black arrows, respectively. Please note that the flow velocities inside the wells are more than a factor of 25 lower than in the channels. c) The E. coli biomass distribution can be visualized by imaging and represented in 3D, indicating that the intensity patterns generally follow the flow velocity profile with high biomass intensities being present in low velocity areas (black arrow), and low biomass in high velocity areas (white arrow). The biomass was measured by trans-UV imaging and the intensity maps were calculated using the ImageJ software.

Article Snippet: ALE on Chip: The lab model bacterium E. coli K12 sub-strain MG1655 DSM 18 039 (DSMZ GmbH, Germany) harboring a kanamycin-selection plasmid (pMK-based backbone, lab stock) was used for all biological experiments.

Techniques: Software, Imaging

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Microfluidic Evolution-On-A-Chip Reveals New Mutations that Cause Antibiotic Resistance.

doi: 10.1002/smll.202007166

Figure Lengend Snippet: Figure 6. Evolution of rifampicin (Rif) resistance in the chip reveals the critical influence of the gradient profile on E. coli adaptability. a) Two con- centration profiles were set as a smooth (chip-A) or steep (chip-B) gradients that reach the same final concentration of 100 µg mL−1 Rif. b) While both gradients showed identical endpoint growth in the chip after 6 days, c) testing of the adapted populations on selective Rif+ agar plates showed the successful evolution of Rif resistance only for cells obtained from the steep gradient chip (plate H). Note that controls plated on the Rif- plates (Figure 6c and Figure S8b, Supporting Information) indicate the presence of living E. coli cells in all samples. For daily images and all well data see Figure S8, Supporting Information.

Article Snippet: ALE on Chip: The lab model bacterium E. coli K12 sub-strain MG1655 DSM 18 039 (DSMZ GmbH, Germany) harboring a kanamycin-selection plasmid (pMK-based backbone, lab stock) was used for all biological experiments.

Techniques: Concentration Assay

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Microfluidic Evolution-On-A-Chip Reveals New Mutations that Cause Antibiotic Resistance.

doi: 10.1002/smll.202007166

Figure Lengend Snippet: Figure 7. Phenotypic and genotypic validation of E. coli mutants adapted to Rif and NA. While the growth of the wild-type E. coli for 24 h is impaired by the presence of Rif or NA, randomly selected mutants from the adaptation experiments to a) Rif and b) NA can grow in selective liquid cultures supplemented with 100 µg mL−1 of Rif and 40 µg mL−1 of NA, respectively. c) Point mutations identified in clones of the adapted E. coli mutants in the resistance-conferring genes: rpoB for Rif and gyrA and gyrB for NA. nt. = position of nucleotide in the gene in relation to the E. coli K12 genome (Gen- Bank: U00096.3). aa. = position of amino acid in the corresponding protein sequence. d) Molecular structure of the E. coli gyrase (PDB ID 6RKW).[58] Two subunits of GyrA (in gray) and two subunits GyrB (in green) form a complex with a DNA molecule. The resistance conferring mutations in the core of the DNA-enzyme complex (red circle) are shown as the wild-type amino acids depicted in red. e) Two NA-resistance mutations (A67S and D82G) are located at the interface of the DNA and two subunits of gyrase A (dark gray). f) Two previously unknown mutations (P747S and Q749P) are located in a loop of gyrase B (green) facing the surface of the neighboring subunit of gyrase A (dark gray, atoms depicted as spheres). All protein structures where generated based on the available crystal structure data (PDB ID 6RKW)[58] using the software PyMOL.

Article Snippet: ALE on Chip: The lab model bacterium E. coli K12 sub-strain MG1655 DSM 18 039 (DSMZ GmbH, Germany) harboring a kanamycin-selection plasmid (pMK-based backbone, lab stock) was used for all biological experiments.

Techniques: Biomarker Discovery, Clone Assay, Sequencing, Generated, Software