DOI: 10.6075/j0542m0k
title: Data from: Allocation of gene products to daughter cells is determined by the age of the mother in single Escherichia coli cells

description: Gene expression and growth rate are highly stochastic in E. coli. Some of
 the growth rate variations result from the deterministic and asymmetric
 partitioning of damage by the mother to its daughters. One daughter,
 denoted the old daughter, receives more damage, grows more slowly, and
 ages. To determine if expressed gene products are also allocated
 asymmetrically, we compared the levels of expressed green fluorescence
 protein in growing daughters descending from the same mother. Our results
 show that old daughters were less fluorescent than new daughters.
 Moreover, old mothers, which were born as old daughters, produced
 daughters that were more asymmetric when compared to new mothers. Thus,
 variation in gene products in a clonal E. coli population also has a
 deterministic component. Because fluorescence levels and growth rates were
 positively correlated, the aging of old daughters appears to result from
 both the presence of both more damage and fewer expressed gene products.
description: <b>Bacterial strains, growth media, and GFP reporter
 </b> Growth experiments were performed using
 <i>E. coli</i> K12 (NCM3722 Δ<i>motA:frt,
 chromosomal:T:ptet</i>-<i>GFP:frt</i>) (43), which has a
 chromosomal insert of constitutively expressed native <i>green
 fluorescent protein</i> (<i>gfp</i>), unfused to any
 protein and thereby with no deterministic spatial placement in the cell as
 a mature protein. Cells were grown in M9 minimal media (44) supplemented
 with 0.02 mg ml<sup>-1</sup> of thiamine, and 0.18 mg
 ml<sup>-1</sup> of glucose as the carbon source. Protein
 levels were quantified by using GFP as a reporter. Because native GFP is estimated to
 have a diffusion rate of 9 μm<sup>2</sup>
 sec<sup>-1</sup>  (31) and <i>E. coli</i>
 cell has a mean cross sectional area of about 3
 μm<sup>2</sup>, the protein is rapidly dispersed throughout a
 cell in less than 1 s. Because our fluorescence images were taken at 20
 min intervals, the distribution of GFP densities in a mother cell, and
 consequently also in the daughters, is not diffusion limited. Rather, the
 different densities result from differential production or gene expression
 within the cells. The strain was kindly provided by Minsu Kim
 (Emory University).   <b>Cell growth and
 microscope slides </b> Cells from -80˚ C glycerol stock
 were streaked onto agar plates. A single colony was inoculated into M9
 media and grown at 37˚ C overnight. The following day the culture was
 diluted 1:100 in M9 and grown for 2 hours. One µl of the culture was then
 pipetted onto a 10 µl M9 agarose pad. The agarose pad was then was then
 flipped with the bacterial side down onto a 24 x 60 mm cover glass and
 placed over a 25 x 75 mm single depression slide sealed with vaseline
 (modified from earlier methods described in (20,21,23) to fit inverted
 microscope). Individual cells from two different movies were followed
 through time lapse microscopy at 37˚ C until each grew into a micro-colony
 of 64 cells. <b>Time-lapse microscopy
 </b> Cells
 were imaged with an inverted microscope (Nikon Eclipse Ti-S), equipped
 with Nikon NIS-Elements AR control software, 100X objective  (CFI Plan APO
 NA 1.4), external phase contrast rings for full intensity fluorescence
 imaging (FITC), fluorescence light source (Prior Lumen 200) with motorized
 shutter (Lambda 10-B Sutter SmartShutter), and camera (Retiga 2000R FAST
 1394, mono, 12 bit). Phase contrast and fluorescence images were recorded
 every 2 and 20 minutes, respectively.
 <b>Image quantification and analysis
 </b> Fluorescence
 measurements were collected by tracing cell outlines on the phase contrast
 images, transferring the outlines to the corresponding fluorescence frame,
 and quantifying density of fluorescence inside the outline. Outlines were
 traced manually. Blind replicate outlines, made without any awareness of
 cell polarity, reproduced the same results. All fluorescence images were
 corrected by removing outliers, subtracting background, and deconvolution
 to correct for diffraction scattering. The software ImageJ (NIH) was used
 for quantifying fluorescence densities, outlier removal, and background
 subtraction.  Fluorescence measurements were collected by first tracing
 cell outlines on the phase contrast images and the corresponding
 fluorescent frame was processed as following: The background of each frame
 was subtracted using "rolling ball" algorithm in ImageJ with
 ball radius 20 pixels. Noise created by heat overflow of single pixel was
 corrected by "remove outliers" algorithm in ImageJ with
 threshold intensity difference 1000 and threshold radius 0.5 pixel. 
 Deconvolution was accomplished by the Lucy-Richardson method in Matlab
 2017b (The MathWorks, Inc., Natick, MA), (see Supplemental Material for
 details). Fluorescence measurements for pairs of old and new daughters
 were normalized by subtracting the mean of the pair’s values. To calculate
 elongation rates, lengths of individual bacterial cells were extracted
 manually from recorded time-lapse images with ImageJ. From lengths
 compiled over time, the elongation rate r was estimated as the slope of a
 linear regression of (log/length) over time. A log transformation was used
 because elongation rates are known to be exponential (20). All lengths
 were measured immediately after division and prior the next
 division. <b>Statistical tests</b> All comparisons were evaluated by either t-tests or randomized designs. Details of sample sizes and choices of paired, unpaired, one- and two-tailed comparisons are provided in the figure legends. Randomized designs were used when data did not conform to standard Gaussian requirements. When appropriate, values are presented as mean ± SEM (standard error of the mean).