All evolved lines and the ancestor were revived from freezer stocks into 2 ml LB and incubated for 12 h with shaking at 250 rpm at 37°C. The cultures were then sub-cultured 1:100 in 2 ml M9 medium, containing 0.2% glycerol as the carbon source, for 12 h. From each tube, cells were then sub-cultured 1:100 in 2 ml M9 glycerol media (a) with and (b) without 0.2% rhamnose, and allowed to grow for 12 h at 250 rpm at 37°C. After growth for 12 h, all lines were sub-cultured to the same initial OD (0.1) into 2 ml M9 glycerol with PQ (40 μM). A volume of 150 μL of these cultures were transferred to a 96-well clear flat-bottom microplate (Costar) in triplicates. The cultures were grown at 37°C in a microplate reader (Tecan Infinite M200 Pro), until they reached stationary phase. OD600 readings were taken every 30 min with 10 min of orbital shaking at 5 mm amplitude before the readings. A gas permeable Breathe-Easy (Sigma-Aldrich) sealing membrane was used to seal the 96-well plates. Growth rate (Malthusian parameter, r) was calculated from the time to reach an OD 1.0, assuming exponential growth in this duration. This growth rate is presented in the growth rates in Figures 4D , 5 .
96 well clear flat bottom microplate
Manufactured by Corning
The 96-well clear flat-bottom microplate is a laboratory equipment designed for various applications in the scientific community. It features a clear, flat-bottom configuration with 96 individual wells, providing a standardized platform for various assays and experiments.
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4 protocols using 96 well clear flat bottom microplate
1
Glycerol-Based Bacterial Growth Kinetics
2
Relative Fitness Assay for MA Lines
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For calculating the relative fitness of the MA lines, cells from the MA lines and the ancestor were revived from the freezer stock in 2ml LB. The cultures were then grown for 12 hours at 37°C and 250 rpm. The cultures were then sub-cultured 1:100 into fresh 2ml LB media. A volume of 150 μL of these cultures were transferred to a 96-well clear flat-bottom microplate (Costar) in triplicates. The cultures were grown at 37°C in an automatic microplate reader (Tecan Infinite M200 Pro), until they reached stationary phase. OD600 readings were taken every 30 minutes with 10 minutes of orbital shaking at 5mm amplitude before the readings. A gas permeable Breathe-Easy (Sigma-Aldrich) sealing membrane was used to seal the 96-well plates. Growth rates were calculated as described in (Mrudula Sane, 2020).
3
FUS Phase Separation under Metabolite Influence
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Assessment of FUS phase separation in different conditions and in the presence of metabolites was performed by turbidity assays. Experiments were carried out by monitoring the OD at 595 nm, using a Benchmark Microplate Reader (Bio‐Rad).
All assays were executed with 5 μM of pure FL FUS in 20 mM Tris–HCl pH 7.00, 20 mM CAPS pH 9.40, and 20 mM CAPS pH 11.00. Exceptions were made when the pH influenced the solubility or stability of the studied metabolites. In particular, the turbidity of FUS solutions in the presence of ZnCl2 was only measured at pH 7.00, since Zn2+ forms an insoluble hydroxide at pH 9.40 and 11.00.
In general, metabolites or RNA solutions in variable concentrations were added to a 96‐flat‐bottom‐well clear microplate (Corning) to a final 100 μl reaction volume. Solutions were subsequently incubated for 60 min at 4°C without agitation. FUS was later added, incubated for additional 10 min and briefly mixed, before the turbidity assays. All measurements were made in triplicate.
All assays were executed with 5 μM of pure FL FUS in 20 mM Tris–HCl pH 7.00, 20 mM CAPS pH 9.40, and 20 mM CAPS pH 11.00. Exceptions were made when the pH influenced the solubility or stability of the studied metabolites. In particular, the turbidity of FUS solutions in the presence of ZnCl2 was only measured at pH 7.00, since Zn2+ forms an insoluble hydroxide at pH 9.40 and 11.00.
In general, metabolites or RNA solutions in variable concentrations were added to a 96‐flat‐bottom‐well clear microplate (Corning) to a final 100 μl reaction volume. Solutions were subsequently incubated for 60 min at 4°C without agitation. FUS was later added, incubated for additional 10 min and briefly mixed, before the turbidity assays. All measurements were made in triplicate.
4
Turbidity Assay for FUS Phase Separation
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Assessment of FUS phase separation in different conditions and in the presence of metabolites was performed by turbidity assays. Experiments were carried out by monitoring the OD at 595 nm, using a Benchmark Microplate Reader (Bio-Rad).
All assays were executed with 5 µM of pure FL FUS in 20 mM Tris-HCl pH 7.00, 20 mM CAPS pH 9.40 and 20 mM CAPS pH 11.00. Exceptions were made when the pH influenced the solubility or stability of the studied metabolites, as previously referred.
In general, metabolites or RNA solutions in variable concentrations were added to a 96-flat-bottom-well clear microplate (Corning) to a final 100 µL reaction volume.
Solutions were subsequently incubated for 60 minutes at 4ºC without agitation. FUS was later added, incubated for additional 10 minutes and briefly mixed, prior to the turbidity assays. All measurements were made in triplicate.
Turbidity of FUS solutions in the presence of ZnCl 2 was only measured at pH 7.00, since Zn 2+ forms an insoluble hydroxide at pH 9.40 and pH 11.00.
All assays were executed with 5 µM of pure FL FUS in 20 mM Tris-HCl pH 7.00, 20 mM CAPS pH 9.40 and 20 mM CAPS pH 11.00. Exceptions were made when the pH influenced the solubility or stability of the studied metabolites, as previously referred.
In general, metabolites or RNA solutions in variable concentrations were added to a 96-flat-bottom-well clear microplate (Corning) to a final 100 µL reaction volume.
Solutions were subsequently incubated for 60 minutes at 4ºC without agitation. FUS was later added, incubated for additional 10 minutes and briefly mixed, prior to the turbidity assays. All measurements were made in triplicate.
Turbidity of FUS solutions in the presence of ZnCl 2 was only measured at pH 7.00, since Zn 2+ forms an insoluble hydroxide at pH 9.40 and pH 11.00.
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