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Fig. 1. Schematic illustration of conventional laboratory approach (A)
and droplet-based microfluidics (B) for tracking the growth of yeast at
population level and the single-cell level, respectively. (C)
Bright-field microscope images of CEN.PK 113-7D growth without organic
acid stress in ~144 pL microdroplets. Scale bar = 50 µm.
Fig. 2. The effect of organic acid stress on the growth of single
wild-type S. cerevisiae (CEN.PK 113-7D) cells in microdroplets
over 24 hours. (A) Plots of the logarithms of the number of cells per
droplets at every hour over the culture of 10 hrs. The μ was calculated
as 0.23 ± 0.03 h-1, and the coefficient of
determination (R2) is 0.97 for the linear fit. (B)
Plots of the number of cells per droplet at eight selected time points:
0 hr, 2 hrs, 4 hrs, 6 hrs, 8 hrs, 10 hrs, 18 hrs and 24 hrs. 60
cell-laden droplets were measured for each time point. The top and
bottom edges of the box refer to the 25th and 75th percentiles, the
cross line represents the median value, the black square represents the
mean value, the whiskers extend to 1.5 times the interquartile range
(IQR) and the asterisks represent upper and lower limits. The
R2 is 0.98 for the fitted growth curve. Scale bar = 50
µm. (C, D) Comparison of the growth of single CEN.PK 113-7D cells at
different concentrations of (C) PA: 0 mM, 7.5 mM and 35 mM, and (D) AA:
0 g/L, 50 mM and 67 mM. 60 cell-laden droplets were measured for each
time point and for each condition.
Fig. 3. The effects of PA and K+ concentration on the
growth of single CEN.PK2-1C cells in microdroplets over 24 hours. (A)
Bright-field and fluorescence microscope images of CEN.PK2-1C growth
without environmental stress in microdroplets at 6 hrs, 10 hrs and 24
hrs. Scale bar = 50 µm. (B) Enlarged bright-field and fluorescence
images showing the growth of CEN.PK2-1C in microdroplets over time.
Scale bar = 50 µm. (C) Plots of the number of cells per droplet at five
selected time points: 2 hrs, 6 hrs, 10 hrs, 18 hrs and 24 hrs. 60
cell-laden droplets for each time point were measured. The top and
bottom edges of the box refer to the 25th and
75th percentiles, the cross line represents the median
value, the black square represents the mean value and the whiskers
extend to 1.5 times the interquartile range (IQR). (D) Comparison of the
growth of single CEN.PK2-1C cells with 25 mA PA and without PA, when the
concentration of K+ is fixed at 10 mM. (E) Comparison
of the growth of single CEN.PK2-1C cells under different concentrations
of K+, 1, 10 and 50 mM, when the concentration of PA
is fixed at 25 mM.
Fig. 4. The growth of wild-type (CEN.PK 113-7D) and PA evolved mutantS. cerevisiae strain (PA-3) in microdroplets over 24 hours when
15 mM PA is applied. 60 cell-laden microdroplets were measured for each
time point. The inset represents the causal mutation for the acquired PA
tolerance identified in PA-3.
Fig. 5. The growth of GFP-tagged P. pastoris strain (CBS7435-GFP)
at the single-cell level in microdroplets over 24 hours. (A)
Bright-field and fluorescence images showing the growth of single
CBS7435-GFP cells in microdroplets over time. Scale bar = 50 µm. (B)
Plots of total fluorescence intensity per droplet. 20 cell-laden
droplets were measured for each time point. The top and bottom edges of
the box refer to the 25th and 75th percentiles, the cross line
represents the median value, the black square represents the mean value,
the whiskers extend to 1.5 times the interquartile range (IQR) and the
asterisks represent upper and lower limits. The insets are bright-field
and fluorescence images of P. pastoris after 24 hours of culture
in microdroplets. Scale bar = 50 µm.
Fig 6. The viability of S. cerevisiae and P. pastorisgrown in the microdroplets experiences no noticeable reduction over 48
hours. (A) Fluorescence images showing the viability of S.
cerevisiae (left) and P. pastoris (right) recovered from
microdroplets after 48 hours of culture. Scale bars are 50 μm for images
obtained by a 20× objective and the insets obtained by a 40× objective.
(B) Bar plots showing cell viability at three time points: 0 hrs (before
encapsulation), 24 hrs and 48 hrs (after encapsulation). Three
repetitions, each of 100 cells, were measured for each time point.