michener jk and smolke cd - epoch life science · michener jk and smolke cd ... supernatants were...

Michener JK and Smolke CD SUPPLEMENTARY INFORMATION Supplementary Figure 1 Decreasing the cell-to-cell variation also decreases the

culture-to-culture variation Supplementary Figure 2 One color fluorescence response histograms Supplementary Figure 3 Screening single cells by FACS is less sensitive than

screening clonal cultures by flow cytometry Supplementary Figure 4 Changing the plasmid copy number does not significantly

affect theophylline accumulation Supplementary Figure 5 Representative HPLC data Supplementary Figure 6 Raw T50 curves Supplementary Figure 7 Plasmid maps Supplementary Table 1 Primers used in this study Supplementary Table 2 Plasmids and strains constructed in this study Supplementary Table 3 Mutations in the BM3 enzyme variants generated in this

study Supplementary Table 4 Summary of functional characterization data for enzyme

variants

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Supplementary Figure 1 Decreasing the cell-to-cell variation also decreases the culture-to-culture variation. When screening by flow cytometry, screening efficiency is determined by the coefficient of variation (CV) between replicate cultures. Expression noise within a single population affects the precision with which the mean can be determined and therefore the CV. For each of the screening constructs shown in Figure 2 (A: plasmid-GAL, B: plasmid-TEF, C: integrated-TEF), replicate cultures (n=32) were grown in the presence of varying amounts of theophylline and the geometric mean fluorescence of each culture was determined by flow cytometry. The relative fluorescence is the ratio of the geometric mean fluorescence for a single culture relative to the average geometric mean for all 32 uninduced cultures. The histograms show raw geometric means and the curves are fit to a normal distribution. The average CV of the three populations decreases from 5.0% (A) to 3.8% (B) and finally to 2.7% (C).

●0.0 mM Theophylline ●0.13 mM Theophylline ●0.27 mM Theophylline

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● 5 mM theophylline● 1 mM theophylline● 1 mM caffeine● Water

● Inactive enzyme, Water● Inactive enzyme, 1 mM caffeine● yCDM1, Water● yCDM1, 1 mM caffeine

● yCDM6, Water● yCDM6, 1 mM caffeine● yCDM1, Water● yCDM1, 1 mM caffeine

A B C

Supplementary Figure 2 One color fluorescence response histograms. (A) Fluorescence histogram of CSY492 grown in the presence of theophylline, caffeine, or water. The screening strain has a graded response to theophylline and no response to caffeine. (B) Fluorescence histograms of CSY492 containing active (green) and inactive (blue) versions of yCDM1. The fluorescence increases when caffeine is added to cells containing the active enzyme but is unchanged when caffeine is added to cells with the inactive enzyme. (C) Fluorescence histograms of CSY492 containing yCDM1 (green) and yCDM6 (blue). The more active enzyme produces a larger increase in fluorescence upon addition of caffeine.

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yCDM1

yCDM3

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yCDM6

yCDM7

yCDM8

Supplementary Figure 3 Screening single cells by FACS is less sensitive than screening clonal cultures by flow cytometry. (A) When screening clonal cultures by flow cytometry, screening efficiency is determined by the coefficient of variation (CV) between replicate cultures. Replicate cultures of cells (n=10) containing the theophylline biosensor were grown in variable amounts of theophylline and analyzed by flow cytometry. The relative fluorescence is calculated as the ratio of the geometric mean fluorescence of a single culture to the average of the geometric mean fluorescence of all ten uninduced cultures. The histograms show raw geometric means, and the curves are fit to a normal distribution. The sensor has a CV of ~2.1%, allowing accurate discrimination of cultures with mean fluorescence differences of <10%. (B) When screening single cells by FACS, the efficiency is determined by the intrinsic noise within a population distribution relative to the difference in population means. Using our optimized sensor, the variation within a population is relatively constant, so the efficiency is strongly dependent on the ratio of fluorescence between the populations being separated. A switch with a larger dynamic range would improve the sorting efficiency, while additional intrinsic noise would reduce the efficiency. Cells containing the two-color screening system were grown in variable amounts of theophylline and analyzed by two-color flow cytometry. A representative sorting gate captured a small fraction of the uninduced cells (<0.1%) and increasing amounts of the more highly fluorescent populations. The ratio of these two percentages gives the predicted enrichment. The relative fluorescence is the ratio of the geometric mean fluorescence of the sample population to the geometric mean fluorescence of the uninduced control. The curve shown is an exponential fit to the experimental data. Screening single cells by FACS is highly effective when the changes in mean fluorescence are large, but this method cannot efficiently discriminate between small changes in mean fluores-cence. (C) Improved enzymes produce more theophylline, which gives a larger fold activation of the RNA switch and, therefore, a better screening efficiency. Theophylline accumulation and switch activation are plotted for each of the enzymes described in this work. The fluorescence change is the increase in fluorescence of cells containing a given enzyme and grown with 1 mM caffeine relative to the same cells grown in the absence of caffeine ([Fcaf-Fwater]/Fwater). The lines are a guide to the eye.

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R2=0.998D

yCDM5High

yCDM6High

yCDM6Low

yCDM7Low

(-)Control

α-V5α-actin

RelativeExpression

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Supplementary Figure 4 Changing the plasmid copy number does not significantly affect enzymatic activity. (A) A Western blot demonstrates that total enzyme expression does not change as the DNA copy number is decreased. The enzyme contains a C-terminal V5 epitope, and the anti-actin antibody is used as a loading control. The relative expression values are calculated as the ratio of anti-V5 intensity/anti-actin intensity, normalized to yCDM6-High. The blot shown is representative of three independent experiments. (B) Total theophylline production differs by less than 10% between yCDM6-High (green) and yCDM6-Low (blue). Theophylline elutes at 0.70 minutes. (C) Fluorescence response histograms for yCDM6-High (green) and yCDM6-Low (blue). For each cell, the GFP fluorescence is normalized by the electronic volume (EV). Despite the similar levels of theophylline production, the low copy expression system shows a smaller change in fluorescence, presumably indicating lower theophylline per cell. (D) Growth curves for cells containing empty plasmid (black), yCDM6-High (green), yCDM6-Low (blue), or an inactive mutant (Neeli et al., 2005) of yCDM1 (brown). The curves are an exponential fit to the data. High-copy enzyme expression causes a significant decrease in growth rate. Lowering the plasmid copy number relieves ~80% of the growth inhibition. While the cells with the low expression system may produce less theophylline per cell, they grow faster and therefore have more time to make theophylline, resulting in similar total production.

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● yCDM1● yCDM3● yCDM5● yCDM6● yCDM7● yCDM8

Theophylline

Paraxanthine

Supplementary Figure 5 Representative HPLC data. Cells containing each enzyme were grown in the presence of 1 mM caffeine. Culture supernatants were analyzed after 24 hours using HPLC analysis. Theophylline elutes at 0.70 minutes and paraxanthine elutes at 0.63 minutes.

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Supplementary Figure 6 Raw T50 curves. Crude E. coli lysate containing each enzyme was incubated for 10 minutes at the indicated temperature and then cooled on ice. 140 µL of enzyme was mixed with 20 µL of 20 mM NADPH and 40 µL of 25 mM caffeine. The residual theophylline production was measured by HPLC and normalized by the activity of lysate incubated at 4 oC. The data were fit to an Arrhenius inactivation curve, and the T50 was calculated as the temperature at which the fit showed 50% residual activity. The error bars show ± one standard deviation, calculated from three technical replicates. Note that these measurements for yCDM5 were an outlier, and replicate measurements did not show a statistically significant difference in T50 between yCDM3 through yCDM8.

pCS21678740 bp

AmpRURA3

yCDM6

ARSH4CEN6

TEFpMB1

ADH1T

EcoRI (2420)

AleI (4558)

Avr II (5696)

Msc I (2718)

Msc I (3434)

pCS21559231 bp

AmpR

URA3yCDM1

TEF2mu

pMB1CYC1 Terminator

EcoRI (445)

AleI (2583)

Msc I (743)

Msc I (1459)

pCS22248404 bp

URA3

EGFP

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mCherry

YIp-Out

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f1 ori

pMB1EcoRI (3239)

BamHI (8164)

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Supplementary Figure 7 Plasmid maps. (A) pCS2223, the single color integration vector used to construct CSY492. (B) pCS2224, the dual color integration vector used to construct CSY820. (C) pCS2172, the E. coli expression vector with yCDM1, used for T50 measurements. (D) pCS2155, the high copy yeast expression vector with yCDM1. (E) pCS2167, the low copy yeast expression vector with yCDM6. (F) The sequence and predicted structure of L2B8 (adapted from Win and Smolke, 2007).

Supplementary Table 1: Primers used in this study

Primer Name Primer Sequence

BM3-FromWori-FWD 5’-TATAGAATTCGATATCAAGCTTGGAGATCTAAAAGAA

AACAATGACAATTAAAGAAATGCCTCAG-3’

BM3-FromWori-REV 5’-CTATGCGGCCGCTCACCCAGCCCACACGTCTTTTG-3’

TEF-FWD 5’-ACTTCTTGCTCATTAGAAAGAAAGC-3’

yCDM-CEN-REV 5’-TATACCTAGGCTTCAATGGTGGTGGTGATGG-3’

yCDM-ToWori-FWD 5’-AATTGGATCCATCGATGCTTAGGAGGTCATATGTCTA

TCAAAGAAATGCCAC-3’

yCDM-ToWori-REV 5’-TAATGAATTCTCAATGGTGGTGGTGATGGTG-3’

yMutF 5’-TCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATC

TAAGTTTTAATTAC-3’

yMutR 5’-AATCTAGCAGTAACTCTGTTGACGATACCTTCGTAGT

TTCTTGGAATAAC-3’

30R 5’-CTTAAAGATTTCACCCAATTCGTCAGCAATTTTCATC-3’

30F 5’-GATGAAAATTGCTGACGAATTGGGTGAAATCTTTAAG-3’

60R 5’-CAAGTTCTTGTCGAATCTAGATTCATCACAAGCTTCC-3’

60F 5’-GGAAGCTTGTGATGAATCTAGATTCGACAAGAACTTG-3’

61R 5’-CAAGTTCTTGTCGAATCTAGATTCATCACAAGC-3’

61F 5’-GCTTGTGATGAATCTAGATTCGACAAGAACTTG-3’

85R 5’-CAGTTCTTTTCGTGGGTCCAGGAAGTGGCCAAACCG-3’

85F 5’-CGGTTTGGCCACTTCCTGGACCCACGAAAAGAACTG-3’

216R 5’-GGAGGCCTTTCTGTCAGCGATGATCTTGTCAAC-3’

216F 5’-GTTGACAAGATCATCGCTGACAGAAAGGCCTCC-3’

329R 5’-GTGTCTTCCTTAGCGTACAAAGAGAACCATGG-3’

329F 5’-CCATGGTTCTCTTTGTACGCTAAGGAAGACAC-3’

341R 5’-CACCCTTTTCCAATGGGTATTCACCACCCAAG-3’

341F 5’-CTTGGGTGGTGAATACCCATTGGAAAAGGGTG-3’

396R 5’-GCGAATTGTTGACCGATACAGGCTCTTTGACCG-3’

396F 5’-CGGTCAAAGAGCCTGTATCGGTCAACAATTCGC-3’

481R 5’-CCATGTTAGAACCGTACAAAACCAACAATG-3’

481F 5’-CATTGTTGGTTTTGTACGGTTCTAACATGG-3’

535R 5’-GCGTTATCTGCTGGATGACCGTTGTAGG-3’

535F 5’-CCTACAACGGTCATCCAGCAGATAACGC-3’

570R 5’-CCCAGTTTTTATCACCA-3’

570F 5’-TGGTGATAAAAACTGGG-3’

586R 5’-CACCCTTAGCAGCCAAAGTTTCGTC-3’

586F 5’-GACGAAACTTTGGCTGCTAAGGGTG-3’

663R 5’-CAATTCCTTGGAGGCAACGACGTTGGTAGAG-3’

663F 5’-CTCTACCAACGTCGTTGCCTCCAAGGAATTG-3’

Supplementary Table 2: Plasmids and strains constructed in this study

Strain Genotype

W303 MATα leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15

CSY492 W303 lys2::PTEF-GFP-L2Bulge8-ADH1T

CSY820 W303 lys2:: PTEF-mCherry-CYC1T-PTEF-GFP-L2Bulge8-ADH1T

pCS1423 Centromeric TRP PTEF-GFP-L2Bulge8-ADH1T

pCS1767 Centromeric URA PGAL-GFP-L2Bulge8-ADH1T

pCS2155 2μ URA PTEF-yCDM1

pCS2156 2μ URA PTEF-yCDM2a

pCS2157 2μ URA PTEF-yCDM2b

pCS2158 2μ URA PTEF-yCDM2c

pCS2159 2μ URA PTEF-yCDM2d


pCS2161 2μ URA PTEF-yCDM4a

pCS2162 2μ URA PTEF-yCDM4b

pCS2163 2μ URA PTEF-yCDM4c

pCS2164 2μ URA PTEF-yCDM4d



pCS2167 Centromeric URA PTEF-yCDM6



pCS2170 2μ URA PTEF-yCDM1 (A264H)

pCS2172 pCWori + yCDM1






pCS2223 pIS385 + Centromeric URA PTEF-yCDM6

pCS2224 pIS385 + PTEF-mCherry-CYC1T-PTEF-GFP-L2Bulge8-ADH1T

CSY821 CSY492+pCS2155









CSY830 CSY492+pCS4 (empty centromeric plasmid)

CSY831 CSY492+pCS31 (empty 2μ plasmid)

CSY845 W303+pCS1767

CSY846 W303+pCS1423

Supplementary Table 3: Mutations in the BM3 enzyme variants generated in this study

Enzyme Mutations

yCDM1 A74W, V78I, A82L, F87A, M185V, L188W, A328F, A330W

yCDM2a yCDM1 + I58T, P461L, A575V

yCDM2b yCDM1 + N522S, C569Y

yCDM2c yCDM1 + T22R, D194N, Q387R, A603T

yCDM2d yCDM1 + S72F, P301L, G457D

yCDM3 yCDM1 + S72F, A603T

yCDM4a yCDM3 + M354L, T576R, Q673K

yCDM4b yCDM3 + F72I, T339I

yCDM4c yCDM3 + R47S

yCDM4d yCDM3 + Q27H, G660D

yCDM5 yCDM3 + Q27H, R47S, F72I

yCDM6 yCDM5 + E435G

yCDM7 yCDM6 + I174V

yCDM8 yCDM7+A87S

Supplementary Table 4: Summary of functional characterization data for enzyme variants

Enzyme Relative

vmax, app/KM,

app

KM, app (mM) T50 (°C) Selectivity

yCDM1 1.0 1.5 ±0.1 47.0 ± 0.6 10.3 ± 0.5

yCDM3 3.9 ±0.4 1.1 ±0.1 42.9 ± 0.9 14 ± 1

yCDM5 12.2 ±1.0 0.75 ±0.10 41.5 ± 1.3 23 ± 3

yCDM6 22.1 ±1.6 0.59 ±0.01 42.8 ± 1.0 100 ±25

yCDM7 26.6 ±3.1 0.74 ±0.02 42.1 ± 1.3 175 ± 4

yCDM8 33.0 ±4.2 0.69 ±0.04 43.1 ± 0.7 230 ±20

michener jk and smolke cd - epoch life science · michener jk and smolke cd ... supernatants were...

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