After the initial evolution experiment, the protocol was modified in an effort to increase the propensity of replicates that were able to survive growth in higher levels of BA. We refer to this as the succeeding evolution experiment; this linguistic choice is twofold as the experiment was the second iteration and was ultimately effective for achieving our goal of continuing to passage a larger number of replicates to a higher level of boric acid. The succeeding evolution experiment was initiated by inoculating the evolved replicates that were frozen at -70 °C during the initial evolution experiment after two transfers in RPMI + 0.8 mg/mL BA (i.e., t4). The freezer culture was thawed and 2 µL of culture was inoculated into 200 µL RPMI + 0.8 mg/mL BA, and
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted January 4, 2024. ; https://doi.org/10.1101/2024.01.04.574193 doi: bioRxiv preprint 8 grown overnight at 37 °C. A single replicate (ancestral strain P87, R6) went extinct after the second transfer of the initial evolution experiment (into 0.4 mg/mL, t2, Figure S1b) and was excluded.
To reduce the extinction rate, we reduced the incremental increase of BA concentration.
After one overnight in RPMI + 0.8 mg/mL BA, surviving replicates were sequentially grown in RPMI + 1.0 mg/mL, 1.25 mg/mL, 1.5 mg/mL, 1.75 mg/mL, 2 mg/mL, 3 mg/mL, and 4 mg/mL BA.
After RPMI + 1.5 mg/mL BA was reached, the number of transfers was changed from two transfers per drug concentration to five transfers. Also after the last transfer in RPMI + 1.5 mg/mL BA, we simultaneously transferred surviving replicates to wells that contained RPMI + 1.75 mg/mL BA and wells that contained RPMI + 2 mg/mL BA. Replicates of five strains (PGC75, P76055, T101, SC5314 and FH1) were unable to grow in RPMI + 2 mg/mL BA within eight days and were classified as group A strains, and the transfer into RPMI + 2 mg/mL BA was discarded. The majority (22 out of 24) of replicates from the other three strains (P87, P78048 and P75016) grew in RPMI + 2 mg/mL BA within eight days and were classified as group B strains, and the transfer into RPMI + 1.75 mg/mL was discarded. As in the initial experiment, cells were frozen down after the last transfer of each drug concentration. No replicates survived growth in RPMI + 4 mg/mL BA. Given the variation among strains in extinction trajectories (i.e., the number of replicates that survived through transfers in different BA concentrations), we chose to focus our phenotypic assessments on a subset of strains. For a balanced design, two strains from each group (Group A: FH1 and GC75; Group B: P75016 and P78048) were chosen. Four ancestral and four evolved replicates isolated from the terminal BA concentration for each strain were examined (FH1: 1.75 mg/mL, GC75: 2 mg/mL, P75016: 3 mg/mL, P78048: 3 mg/mL; Table 1).
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted January 4, 2024. ; https://doi.org/10.1101/2024.01.04.574193 doi: bioRxiv preprint 9
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted January 4, 2024. ; https://doi.org/10.1101/2024.01.04.574193 doi: bioRxiv preprint 8 grown overnight at 37 °C. A single replicate (ancestral strain P87, R6) went extinct after the second transfer of the initial evolution experiment (into 0.4 mg/mL, t2, Figure S1b) and was excluded.
To reduce the extinction rate, we reduced the incremental increase of BA concentration.
After one overnight in RPMI + 0.8 mg/mL BA, surviving replicates were sequentially grown in RPMI + 1.0 mg/mL, 1.25 mg/mL, 1.5 mg/mL, 1.75 mg/mL, 2 mg/mL, 3 mg/mL, and 4 mg/mL BA.
After RPMI + 1.5 mg/mL BA was reached, the number of transfers was changed from two transfers per drug concentration to five transfers. Also after the last transfer in RPMI + 1.5 mg/mL BA, we simultaneously transferred surviving replicates to wells that contained RPMI + 1.75 mg/mL BA and wells that contained RPMI + 2 mg/mL BA. Replicates of five strains (PGC75, P76055, T101, SC5314 and FH1) were unable to grow in RPMI + 2 mg/mL BA within eight days and were classified as group A strains, and the transfer into RPMI + 2 mg/mL BA was discarded. The majority (22 out of 24) of replicates from the other three strains (P87, P78048 and P75016) grew in RPMI + 2 mg/mL BA within eight days and were classified as group B strains, and the transfer into RPMI + 1.75 mg/mL was discarded. As in the initial experiment, cells were frozen down after the last transfer of each drug concentration. No replicates survived growth in RPMI + 4 mg/mL BA. Given the variation among strains in extinction trajectories (i.e., the number of replicates that survived through transfers in different BA concentrations), we chose to focus our phenotypic assessments on a subset of strains. For a balanced design, two strains from each group (Group A: FH1 and GC75; Group B: P75016 and P78048) were chosen. Four ancestral and four evolved replicates isolated from the terminal BA concentration for each strain were examined (FH1: 1.75 mg/mL, GC75: 2 mg/mL, P75016: 3 mg/mL, P78048: 3 mg/mL; Table 1).
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted January 4, 2024. ; https://doi.org/10.1101/2024.01.04.574193 doi: bioRxiv preprint 9
