real-time multivariate detection from single cells monitoring the metabolism of methylobacterium...
TRANSCRIPT
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Real-Time Multivariate Detection from Single Cells
Monitoring the Metabolism of Methylobacterium extorquens
AM1
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Overview
Microscale Life Science Center Methylobacterium extorquens AM1 Green Fluorescent Protein (GFP) as a
transcriptional reporter Detection of respiration rates Multi-variate detection of single cells
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MLSC
Funded by NIH CEGS To develop technologies for single cell
research Lab-on-a-chip modality
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Why Single Cells?
Variable of interest Bulk data represents
averages Averages may not
represent behavior of subpopulations
1 2 3 4 5 6 7
Singular Resonse50% response
Range of Response0
1
2
3
4
5
6
7
8
9
10
Intensity of Response
Potential Resonse Profiles for a Population
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Methylobacterium extorquens AM1
Gram- bacterium (like E. coli) Capable of growing on
methanol and multicarbon substrates (succinate)
Industrial interest for production of value added products
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periplasm
cytoplasm
MeOH
Methylotrophic Metabolism
Formaldehyde
Central Metabolism
(Methanol Dehydrogenase)
(Formaldehyde Activating Enzyme)
(Carbon Assimilation)
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Goals
Hypothesis: Behavior of single cells differ from that of
averaged populations Approach:
Develop and utilize technology to study single cells
Characterize single cells in contrast to populations
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Populations to Single Cells
Use GFP as a reporter of transcriptional activity Will reflect promoter activity
Observed GFP fluorescence during growth on methanol and succinate Observe in bulk and at the single cell level
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Green Fluorescent Protein
HO
O
N
N
N
OH
O
BA
HO
O
N
N
N
OH
O
HO
O
N
N
N
OH
HO
O
N
N
HOHO
O
N
N
N
OH
O
BA
First isolated from Aequorea victoria Emits fluorescence at 509nm
Coral is another source for many color variants
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Genetic ManipulationSuicide Vector
Chromosome
Chromosome
KanR GFPuv
Double Crossover Event
Red regions = homologous sequence
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Genetic Fusions
PMDH GFPuv
Transcriptional Fusion
•Methanol Growth Higher GFP expression•Succinate Growth Lower GFP expression
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FluorimetryGFPuv Accumulation During Growth
0
50
100
150
200
250
300
350
0.2 0.3 0.4 0.5 0.6 0.7 0.8
OD600nm
RFU
(509
nm)
MethanolSuccinate
Strovas et al. In preparation.
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Data can be used for the calculation of promoter activities
Is a gauge of gene transcription in bulk culture
Promoter activity dictated by multiple variables
Calculating Promoter Activities
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Equations for Modeling Promoter Activity
Leveau and Lindow, 2001
Non-fluorescent FP (n)
Fluorescent FP (f)
Dilution from Cell Division
Degradation
MaturationSynthesis
P m n
n f
Vmax nn + f + KM
Vmax fn + f + KM
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Establish RFU/O.D. 600nm plot P = fss*(1 + /m)
fss = RFU/OD600nm
= generation time m = maturation rate of GFP
Units are RLU/OD600nm*hr
Equations for Modeling Promoter Activity
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FluorimetryGFPuv Accumulation During Growth
0
50
100
150
200
250
300
350
0.2 0.3 0.4 0.5 0.6 0.7 0.8
OD600nm
RFU
(509
nm)
MethanolSuccinate
Strovas et al. In preparation.
349.1 +/- 82.59
264.3 +/- 10.27
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Single Cell Growth Assays
Observed growth of single cells
Determined divisions rates
Measured fluorescence content
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Single Cell Growth Assays
Video using LSM software
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LSM Experiments
Single Cell Growth Profile
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
0 5 10 15 20
Time (hrs)
Cel
l Len
gth
( m
)
Strovas et al. In preparation.
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LSM Experiments
Single Cell Growth Profile
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
0 5 10 15 20
Time (hrs)
Cel
l Len
gth
( m
)
Strovas et al. In preparation.
0.55m/hr
0.73 m/hr
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LSM Experiments
Division Times for Growth on Succinate
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101
106
111
Tim
e (h
rs)
Distribution of Division Times During Growth on Succinate
0
2
4
6
8
10
12
14
16
18
20
Time (hrs)
Fre
qu
ency
Strovas et al. In preparation.
3.12 +/- 0.55 hrs (N = 115)
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LSM Experiments
Divisions Times During Methanol Growth
0
1
2
3
4
5
6
7
1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161 171 181 191
Tim
e (h
rs)
Distribution of Division Times During Growth on Methanol
0
5
10
15
20
25
Time (hrs)
Fre
qu
ency
Strovas et al. In preparation.
3.73 +/- 0.63 hrs (N = 195)
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LSM ExperimentsSingle Cell Growth on Succinate
Strovas et al. In preparation.
1400
1500
1600
1700
1800
1900
2000
2100
2200
0 0.2 0.4 0.6 0.8 1 1.2
Growth Rate (m/hr)
RF
U
1400
1500
1600
1700
1800
1900
2000
2100
2200
1.5 2 2.5 3 3.5 4 4.5 5
Area (m^2)
RFU
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LSM ExperimentsSingle Cell Growth on Methanol
Strovas et al. In preparation.
1800
1900
2000
2100
2200
2300
2400
2500
0 0.2 0.4 0.6 0.8 1 1.2
m^2/hr
RF
U
1800
1900
2000
2100
2200
2300
2400
2500
1.5 2 2.5 3 3.5 4 4.5
Area (m^2)
RF
U
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LSM Experiments
Succinate -> MeOH
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
0 5 10 15 20 25 30 35
Time (hrs)
Sin
gle
Cel
l RF
U/
m^2
Methanol -> Succinate
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
0 5 10 15 20 25 30 35 40
Time (hrs)
Sin
gle
Cel
l RF
U/
m^2
Single Cell Carbon Shifts
Succinate: 1993.15 +/- 468.14 RFU/m^2 (N = ~1000)Methanol: 3075.30 +/- 243.35 RFU/m^2 (N = ~1000)
Strovas et al. In preparation.
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GFPuv is a viable reporter in M. extorquens AM1
Data averages obscure subpopulation dynamics
Populations to Single Cells
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Measuring Respiration Rates
Measured respiration rates from bulk cultures of M. extorquens AM1
Utilized Pt-porphyrin doped beads that are an inverse sensor of [O2]
Signals acquired are phosphorescent lifetimes
Samples and beads were sealed in 4ml cuvette and monitored over time
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Fluorescence Phosphorescence
Intersystemcrossing
Absorption
Quenching
O2
Ene
rgy
Singlet Excited State
TripletExcited State
Bulk Respiration rates
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Light Dark
Io(1 – e-Kt) Ioe-Kt
a
b
Log(b/a) = Lifetime of decay
Bulk Respiration rates
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y = 5160.2x + 1.0671
R2 = 0.9978
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.0001 0.0002 0.0003 0.0004 0.0005
Mol O/L
To /
T
0
10
20
30
40
50
60
70
Life
times
(s)
Bulk Respiration rates
Strovas and Dragavon et al. J. Environ Microbiol. (accepted)
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Bulk Respiration rates
Strovas and Dragavon et al. J. Environ Microbiol. (accepted)
B
0
0.0001
0.0002
0.0003
0.0004
0.0005
0.0006
0 10 20 30 40 50 60Time (min)
Mol
O/L
A
0
10
20
30
40
50
60
0 10 20 30 40 50 60Time (min)
Lif
etim
es (
sec)
Respiration rate (Mol O/min*cell e-17) Methanol = 5.4 +/- 0.74
Succinate = 3.8 +/- 0.89
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Multi-variate detection from single cells
Utilize multiple fluorescent proteins as transcriptional probes
Measure respiration rates as a gauge of metabolic activity and cell health
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Methylotrophic Metabolism
GFP
YFP
RFP
Methanol Oxidation
Formaldehyde Oxidation
Carbon Assimilation
Central Metabolism
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Current Approach
Aqueous phaseHydrophobic Phase
Hydrophobic Phase
Oil water separation for spatial isolationUtilize 50-100m square capillariesUse free floating porphyrin beads
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Oil and Water
250m capillary4nL aqueous volumes
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End Goals
Achieve single respiration rate detection Measure gene expression in single cells with
three fluorescent proteins Use all four measurements as a
comprehensive analysis of M. extorquens AM1 response to growth on methanol and succinate
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Acknowledgements
Dr. Mary Lidstrom MLSC The Lidstrom Lab
Dr. Joseph Chao Dr. Mark Holl Joe Dragavon Tim Molter Cody Young Linda Sauter Tylor Hankins Angela Burnside