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Saturday, November 8 iGEM Jamboree
Katherine Jacobs Neil Parikh Rima Shah
Brown iGEM Lab
'DrinkingwaterintheU.S.isamongthetopfourpublichealthrisksposedbyenvironmentalproblems.'‐‐FormerEPAAdministratorWilliamK.Reilly
Accordingtosomeestimates,arsenicindrinking‐waterwillcause200,000–270,000deathsfromcancerinBangladeshalone.‐‐NRC,1998;Smith,etal,2000
Fieldtestkitscandetecthighlevelsofarsenicbutaretypicallyunreliableatlowerconcentrationsofconcernforhumanhealth.Reliabilityoffieldmethodsisyettobefullyevaluated.
‐Guidelinesfordrinking‐waterquality,2nded. Geneva,WorldHealthOrganization,1996.
Bulky Laboratory setting
Biological
Uses minimal biological machinery Direct induction of system allows for sensitive measurements Versatile construct
Engineering
Compact and user-friendly system for sample analysis Economically feasible
Biological
Cell Lysis Cassette under the control of an inducible promoter ▪ Potential promoters: Arsenic, Lead, and Mercury ▪ Proof of Concept: pBAD (Arabinose Induced) promoter
Electrical
Cell Lysis will cause a release of ions Change in resistance/conductance of bacterial solution
Objective: To detect the presence of heavy metals in a water sample.
Inducible promoter (pBAD = proof of concept)
RS
S
R
Holin
Endolysin
Rz Function Not Characterized
Rz RS Lysis Cassette BBa_K124003 / BBa_K124014
BBa_K124017
In Progress
RS
S R
II I
I
S, R, Rz proteins are produced
S
R S
R
R
S
R
S
Let’s take a closer look…
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S S
Outside Cell
Inside Cell
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The S proteins channel through the inner membrane
R
The R proteins, endolysins, then enter the membrane and initiate lysis
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The cell wall degrades & fragments
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Ions leak out of cell
Figure 1: Change in optical density before and after lysis, standardized to the control.
Figure2:Visualchangeinopticaldensity
Decrease in optical density occurred several hours after induction with Arabinose.
But how long does lysis really take?
Control
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ical
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sity
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nm)
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ical
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sity
(600
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Test
After Lysis Control
Before Lysis
Test Control
Change in resistance should correlate to change in optical density
Control : (-Ara) Test: (+Ara)
Decrease in OD indicates lysis of cells (+Ara)
Biological
Cell Lysis Cassette under the control of an inducible promoter ▪ Potential promoters: Arsenic, Lead, and Mercury ▪ Proof of Concept: pBAD (Arabinose Induced) promoter
Electrical
Cell Lysis will cause a release of ions Change in resistance/conductance of bacterial solution
Objective: To detect the presence of heavy metals in a water sample.
Design V1
Design V2
Design V3
Considerations: -Alternating Current -Electrode Material -Fixed Electrodes
Hypothesis Resistance of concentrated solution will decrease after lysis.
Conclusion Change seen but system noise = sensitivity
Resistance of 50x Concentrated Cell Solution Before & After Lysis
Vernier Go!Link Conductivity Probe
Sensitivity
Commercially Available
Alternating Current
Graphite Electrodes
Fixed electrodes
Calibration
Hypothesis: Conductivity will increase during lysis.
Conclusion: Conductivity increase correlates to optical density decrease as cellular lysis occurs.
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Cond
ucta
nce
(uS/
cm)
Time (Hours)
Test Control -- Predicted
Conductance Measurements of Cell Lysis Over Time
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14300
14500
14700
14900
15100
15300
15500
0 2 4 6 8 10 12 14 16 18 20 22 24
Cond
ucta
nce
(uS/
cm)
Time (Hours)
Test Control
Conductance Measurements of Cell Lysis Over Time Optical Density Measurements of Cell Lysis Over Time
Our Design
Biological
Uses minimal biological machinery Direct induction of system by inducer creates a sensitive system Versatile construct
Engineering
Compact and user-friendly system for sample analysis Economically feasible
Electrical & Computing
Based System
Conductance Measuring Circuit
VS.
<$1.00 $5,000+
Synthetic Biology
- Potential alternative to Biological Reporters i.e. GFP
“There is a need for an effective bacterial kill switch, especially for outer space applications, as we introduce organisms into new environments.”
-John Cumbers [NASA AMES]
Auto-lysis of cells can protect against uncontrollable protein expression
Machinery designed to be incorporated into future SynBio systems
ProfessorsGaryWessel,TayhasPalmore,JerryDaniels,AlexBrodsky,CarlosAizenman‐Stern,andJillKreiling
GraduateStudentsAdrianReich,DianaDonovan,JamieGagnon,VinceSiu
DanielLudwig,GaurabChakrabarti JohnCumbers,AdellaFrancis DeepaGalaiya,JeffHoffman,TitoJankowski
Questions?
WorldHealthOrganizationFactSheetHealthinWaterResourcesDevelopment. WHO,2008.Saferwater,betterhealth. UNICEF/WHO.2008.ProgressonDrinkingWaterandSanitation:SpecialFocusonSanitation. "UseofLambdaPhageSandRGeneProductsinanInducibleLysisSystemforVibriocholerae‐and
SalmonellaentericaSerovarTyphimurium‐BasedDNAVaccineDeliverySystems"VIVEKJAINANDJOHNJ.MEKALANOS,HarvardMedicalSchool
“MutationalanalysisofbacteriophagelambdalysisgeneS”,RRAAB,GNEAL,JGARRETT,RGRIMAILA,RFUSSELMAN,ANDRYOUNG
“DevelopmentofaSimpleCellLysisMethodforRecombinantDNAUsingBacteriophageLamdaLysisGenes”,JANG,BOYUN,YUNAJUNG,ANDDONGBINLIM
“Celllysisbyinductionofclonedlambdalysisgenes”‐GARRETTJ,FUSSELMANR,HISEJ,CHIOUL,SMITH‐GRILLOD,SCHULZJ,YOUNGR
E.coliInformationDatabase:InstituteforBiomolecularDesign,UniversityofCalgary “PhiX174EcomplementslambdaSandRdysfunctionforhostcelllysis”‐WDROOFANDRYOUNG USpatent(100/17300)MethodtoEliminateTransientSpikesonFluidicFlowCausedbyInadequateCurrent
andVoltageChanges,byCaliperLifeSciences(2002) "HolinsKillWithoutWarning"PNASANGELIKAGRUNDLING,MICHAELD.MANSON,RYYOUNG "HOLINS:TheProteinClocksofBacteriophageInfections"AnnualReviews:ANonprofitScientificPublisher‐
http://redpoll.pharmacy.ualberta.ca/CCDB/cgi‐bin/STAT_NEW.cgi
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