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

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S, R, Rz proteins are produced

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R S

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R

S

Let’s take a closer look…

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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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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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Test Control -- Predicted

Conductance Measurements of Cell Lysis Over Time

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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