Fig. 5: Effect of macromolecular crowding and transcription on conformation of protein-free chromosomes.

a Scheme: protein-free chromosomes by on-chip cell lysis and protein degradation with protease Proteinase K (PKA). Right panels: exemplary time-lapse montages of E. coli cells labeled through RNAP (β’-HT) undergoing lysis (between 80-100 s) with (lower row) and without (upper row) protein degradation by PKA to obtain protein-free and protein-bound chromosomes, respectively. b Exemplary snapshots of SYBR Green I (SG-I) labeled chromosomes after transplantation and protein degradation with PKA at different PEG concentrations in dilute buffer, in a TxTl system, and in a TxTl system with 500 nM rifampicin (rif) to inhibit transcription initiation. c Chromosome areas were estimated in buffer conditions from 4 independent biological experiments at 4.0% PEG (N = 12 chromosomes analyzed), 7.0% (N = 105), 9.0% (N = 298), and 10.0% (N = 151). The TxTl system was analyzed from 3 independent biological experiments at 1.0% PEG (N = 321), 2.0% (N = 28), 2.5% (N = 122), 3.5% (N = 120), 4.5% (N = 173), and 7.0% (N = 90). The TxTl system with rif was analyzed from 2 independent biological experiments at 0.0% PEG (N = 146), 1.0% (N = 318), 2.0% (N = 184), and 3.6% (N = 139), where N is the total number of chromosomes analyzed. The graph shows the means of the population for the three different conditions, with error bars showing +/− 84.1% and 15.9% quantiles. d Exemplary snapshots of a region inside a large compartment populated with SG-I labeled chromosomes in a TxTl system undergoing a reversible transition from open (2.5% PEG) to compact (4.5% PEG) and back (1.5% PEG) with a change in PEG concentrations. Chromosomes were incubated with continuous imaging for ~20 minutes for each condition. The reversibility was checked in at least two independent biological experiments. e Estimated areas of chromosomes incubated in the three indicated conditions and labeled with SG-I (same data as in e). Individual chromosomes are shown as the smaller gray points. The boxplot extends from the first quartile (25%) to the third quartile (75%) of the data, with a line at the median and a circle at the mean. The whiskers extend from the box to the farthest data point lying within 1.5-fold the inter-quartile range from the box. Two-sided Mann-Whitney U tests were performed to compute the statistical significance between the distributions. The same statistics from c apply here. f The estimated summed area of all chromosomes in a large compartment was tracked during the transition from active to inactive TxTl (2% PEG), controlled with 500 nM rif (diluted from stock dissolved in DMSO). The black curve shows raw data after image processing, and the yellow curve shows data smoothed with a Gaussian kernel, both normalized by the initial area at the start of the experiment. The areas of 105 chromosomes were analyzed in an experiment. g Chromosomes from g were again exposed to a TxTl system (2% PEG) without rif (instead, DMSO was added at the same dilution level as in g). The overall chromosome area estimate is shown as the black curve and smoothed by a Gaussian kernel shown as the yellow curve, both normalized by the initial area at the start of the experiment. The areas of 123 chromosomes were analyzed in an experiment. Source data is provided as a Source Data file.