cornell university genetically engineered machines2009.igem.org/files/presentation/cornell.pdf–dr....
TRANSCRIPT
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Cadmium Poisoning• Ingestion of cadmium can cause:
– Cancer– Renal failure– Bone weakening and fracture– Damage to nervous and immune
systems– Death
• Danger Level– Present in over 60% of hazardous
waste sites as identified by the EPA
http://ic.ucsc.edu/~flegal/etox80e/Images/itai-itai.jpg
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Sources of contamination
• Mining operations
• Sewage
• Irrigation water
http://farm2.static.flickr.com/1038/861723910_888a1dcdb9.jpg
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Cadmium Detection
• Current cadmium measurement techniques
– Atomic Absorption Spectroscopy
• Advantages of Biosensor
– More affordable
– Measures bio-available cadmium
http://meri.njmeadowlands.gov/lab/Images/Varian%20AA220.JPG
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• Engineer B. subtilis to sense cadmiumObjective
Create BiobrickParts
Construct Devices
Transform Devices into B. subtilis
Characterize Chassis
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Chassis
Bacillus subtilis• Gram positive soil bacteria
• Well characterized
• Naturally competent
• Complex metal ion homeostasis system
http://www.nasa.gov/images/content/177389main_POEMS1.jpg:
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Metal Ion Homeostasis
Efflux
Protein
(CadA)
Influx
Protein
(MntH)
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Inactive Repressor (MntR)
Cadmium Influx Regulation
Repressor
(mntR)
Cadmium
Repressor
(mntR)
Active Repressor (MntR)
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Low Intracellular [Cadmium]
Influx
Protein
(MntH)
Influx Protein Gene (mntH)
Influx Promoter
(MntH)
Cadmium Inactive
Repressor
(MntR)
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High Intracellular [Cadmium]
Influx Protein Gene (mntH)
Influx Promoter
(MntH)
Cadmium
Active
Repressor
(MntR)
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Inactive Repressor (czrA)
Cadmium Efflux Regulation
Repressor (mntR)
Cadmium
Repressor (mntR)
Active Repressor (czrA)
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Low Intracellular [Cadmium]
Efflux Protein Gene (cadA)
Efflux Protein (CadA)
Promoter
Cadmium
Active
Repressor
(CzrA)
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High Intracellular [Cadmium]
Efflux Protein Gene (cadA)
Efflux Protein (CadA)
Promoter
CadmiumInactive
Repressor
(CzrA)
Efflux
Protein
(CadA)
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• Engineer B. subtilis to sense cadmiumObjective
• Influx Protein (mntH)
• Efflux Protein (cadA)
• Internal Reference (mrgA, mrgArbs)
Create BiobrickParts
• Cadmium Sensor Module A
• Cadmium Sensor Module B
• Internal Reference ModuleConstruct Devices
Transform Devices into B. subtilis
Characterizing Chassis
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Cadmium Sensor Module A
• Absence of cadmium increases expression of YFP reporter sequence
RBS YFP Coding Sequence
Influx Promoter (MntH)
YFP
Inactive
Repressor
(MntR)
No Repression
Expression
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Influx Promoter(MntH)
Repression
Active
Repressor
(MntR)
Inactive
Repressor
(MntR)Cd(II)
No Expression
Module A: Active Form
RBS YFP Coding Sequence
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Cadmium Sensor Module B
• Absence of cadmium decreases expression of CFP reporter sequence
RBS CFP Coding Sequence
Efflux Promoter(CadA)
Active
Repressor
(CzrA)
Repression
No Expression
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Cadmium
Active
Repressor
(CzrA)
Inactive
Repressor
(CzrA)
No Repression
RBS CFP Coding Sequence
Efflux Promoter(CadA)
CFPExpression
Module B: Active Form
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Possible outputs
Condition Module A (influx – mntH)
Module B(efflux – cadA)
Detectable Cadmium
UndetectableCadmium
Dead cells
Legend
Active
Inactive
Can’t distinguish between an undetectable level of cadmium
and dead cells!
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Internal Reference Module
• (Mutated) Metal regulated Gene A (mrgA) reference promoter
• Constitutive
• Similar level of expression between mrgA and cadA promoters
Reference Promoter(mrgA)
RBS GFP Coding Sequence
GFPExpression
Constitutive
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New possible outputs
Condition Module A(influx – mntH)
Module B(efflux – cadA)
Internal ReferenceModule
SufficientCadmium
InsufficientCadmium
Dead Cells
Can distinguish between conditions!
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Integrated System
RBS CFP Coding Sequence
RBS YFP Coding Sequence
RBS GFP Coding Sequence
Module A (Influx protein – mntH)
Module B (Efflux protein – cadA)
Internal Reference Module
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• Engineer B. subtilis to sense Cd(II)Objective
• Influx protein (mntH)
• Efflux protein (cadA)
• Internal Reference(mrgA, mrgA rbs)Create Biobrick Parts
• Cadmium Sensor Module A
• Cadmium Sensor Module B
• Reference ModuleConstruct Devices
• Single Homologous Recombination
• Double Homologous RecombinationTransform Devices into B. subtilis
Identify Appropriate Strain for Chassis
*Intermediate Constructs Completed
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Integration into B. subtilisChromosomal DNA
• Single Homologous Recombination
Plasmid
Chromosome
RBS CFP Coding Sequence
Efflux Promoter(CadA)
Efflux Protein Gene (cadA)
Efflux Promoter (CadA)
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Integration into B. subtilisChromosomal DNA
• Double Homologous Recombination
amyE
Plasmid
Chromosome
Reference Promoter(mrgA)
RBS GFP Coding Sequence5’ amyE 3’ amyE
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• Engineer B. subtilis to sense Cd(II)Objective
• Influx protein (mntH)
• Efflux protein (cadA)
• Internal Reference(mrgA, mrgA rbs)
Create BiobrickParts
• Cadmium Sensor Module A
• Cadmium Sensor Module B
• Reference ModuleConstruct Devices
• Single Homologous Recombination
• Double Homologous RecombinationTransform Devices into B. subtilis
• WT
• mntH null
• mntR null
Characterizing for Chassis
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Characterizing Chassis: WT
• Need to find appropriate sensing range
0 uM
2.5 uM
5 uM
10 uM
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Optimizing Sensitivity
• Increase number of healthy cells
• Increase sensitivity of each cell
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Characterizing Chassis: mntH null
• mntH null growth normal at high [Cadmium]
mntH null
wild type
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Characterizing Chassis: mntR null
Influx Protein Gene (mntH)
Influx Promoter (MntH)
Cd(II)
No MntR
Repressor
Influx
Protein
(MntH)
• mntR null increases sensitivity to Cadmium
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Optimizing Sensitivity
• Increase number of healthy cells
– Decrease Cadmium influx by reducing mntRexpression
• Increase sensitivity of each cell
– Increase Cadmium influx by reducing mntH
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• Engineer B. subtilis to sense Cd(II)Objective
• Influx protein (mntH)
• Efflux protein (cadA)
• Internal Reference(mrgA, mrgA rbs)
Create BiobrickParts
• Cadmium Sensor Module A
• Cadmium Sensor Module B
• Reference ModuleConstruct Devices
• Single Homologous Recombination
• Double Homologous RecombinationTransform Devices into B. subtilis
• WT
• mntH null
• mntR null
Characterizing for Chassis
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Future Work
• Complete Cadmium Sensor Modules
• Transform constructs in B. subtilis
• Compare cadmium sensitivity in different chassis: WT, Influx protein (mntH), Influx protein represssor (mntR) tuned strains
• Additional metal sensing promoters: copper(copZ),arsenic(arsR)
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Advisors and Sponsors• Advisors:
– Dr. Maki Inada– Dr. Carl Batt– Dr. John D. Helmann– Dr. Xiling Shen
• Sponsors:– College of Engineering– Weill Institute of Cell and Molecular Biology– College of Agriculture and Life Sciences– SAFC– Bartels Co-sponsorship
• Special thanks to: – Dr. Jeff Pleiss and his lab– Dr. Ahmed Gaballa– Dr. Kent Kemphues