Real-Time Multivariate Detection from Single Cells
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Transcript of Real-Time Multivariate Detection from Single Cells
Real-Time Multivariate Detection from Single Cells
Monitoring the Metabolism of Methylobacterium extorquens
AM1
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
MLSC
Funded by NIH CEGS To develop technologies for single cell
research Lab-on-a-chip modality
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
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Intensity of Response
Potential Resonse Profiles for a Population
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
periplasm
cytoplasm
MeOH
Methylotrophic Metabolism
Formaldehyde
Central Metabolism
(Methanol Dehydrogenase)
(Formaldehyde Activating Enzyme)
(Carbon Assimilation)
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
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
Green Fluorescent Protein
HO
O
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N
OH
O
BA
HO
O
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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
Genetic ManipulationSuicide Vector
Chromosome
Chromosome
KanR GFPuv
Double Crossover Event
Red regions = homologous sequence
Genetic Fusions
PMDH GFPuv
Transcriptional Fusion
•Methanol Growth Higher GFP expression•Succinate Growth Lower GFP expression
FluorimetryGFPuv Accumulation During Growth
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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.
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
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
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
FluorimetryGFPuv Accumulation During Growth
0
50
100
150
200
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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
Single Cell Growth Assays
Observed growth of single cells
Determined divisions rates
Measured fluorescence content
Single Cell Growth Assays
Video using LSM software
LSM Experiments
Single Cell Growth Profile
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0 5 10 15 20
Time (hrs)
Cel
l Len
gth
( m
)
Strovas et al. In preparation.
LSM Experiments
Single Cell Growth Profile
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Time (hrs)
Cel
l Len
gth
( m
)
Strovas et al. In preparation.
0.55m/hr
0.73 m/hr
LSM Experiments
Division Times for Growth on Succinate
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Tim
e (h
rs)
Distribution of Division Times During Growth on Succinate
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Time (hrs)
Fre
qu
ency
Strovas et al. In preparation.
3.12 +/- 0.55 hrs (N = 115)
LSM Experiments
Divisions Times During Methanol Growth
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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
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25
Time (hrs)
Fre
qu
ency
Strovas et al. In preparation.
3.73 +/- 0.63 hrs (N = 195)
LSM ExperimentsSingle Cell Growth on Succinate
Strovas et al. In preparation.
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0 0.2 0.4 0.6 0.8 1 1.2
Growth Rate (m/hr)
RF
U
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Area (m^2)
RFU
LSM ExperimentsSingle Cell Growth on Methanol
Strovas et al. In preparation.
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0 0.2 0.4 0.6 0.8 1 1.2
m^2/hr
RF
U
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Area (m^2)
RF
U
LSM Experiments
Succinate -> MeOH
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0 5 10 15 20 25 30 35
Time (hrs)
Sin
gle
Cel
l RF
U/
m^2
Methanol -> Succinate
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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.
GFPuv is a viable reporter in M. extorquens AM1
Data averages obscure subpopulation dynamics
Populations to Single Cells
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
Fluorescence Phosphorescence
Intersystemcrossing
Absorption
Quenching
O2
Ene
rgy
Singlet Excited State
TripletExcited State
Bulk Respiration rates
Light Dark
Io(1 – e-Kt) Ioe-Kt
a
b
Log(b/a) = Lifetime of decay
Bulk Respiration rates
y = 5160.2x + 1.0671
R2 = 0.9978
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0 0.0001 0.0002 0.0003 0.0004 0.0005
Mol O/L
To /
T
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Life
times
(s)
Bulk Respiration rates
Strovas and Dragavon et al. J. Environ Microbiol. (accepted)
Bulk Respiration rates
Strovas and Dragavon et al. J. Environ Microbiol. (accepted)
B
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0 10 20 30 40 50 60Time (min)
Mol
O/L
A
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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
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
Methylotrophic Metabolism
GFP
YFP
RFP
Methanol Oxidation
Formaldehyde Oxidation
Carbon Assimilation
Central Metabolism
Current Approach
Aqueous phaseHydrophobic Phase
Hydrophobic Phase
Oil water separation for spatial isolationUtilize 50-100m square capillariesUse free floating porphyrin beads
Oil and Water
250m capillary4nL aqueous volumes
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
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