FRAGRANCE- PRODUCING CO 2015 LASATX iGEM SENSOR RATIONALE AND - - PowerPoint PPT Presentation

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FRAGRANCE- PRODUCING CO 2015 LASATX iGEM SENSOR RATIONALE AND - - PowerPoint PPT Presentation

Sep 07, 2022 30 likes •286 views

FRAGRANCE- PRODUCING CO 2015 LASATX iGEM SENSOR RATIONALE AND HUMAN PRACTICES/DESIGN Disabled Our project: Fragrance Natural disasters Power outages Energy-independent Reliance on gasoline generators Transportable

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SLIDE 1

FRAGRANCE- PRODUCING CO SENSOR

2015 LASATX iGEM

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SLIDE 2

RATIONALE AND HUMAN PRACTICES/DESIGN

  • Disabled
  • Natural disasters
  • Power outages
  • Reliance on gasoline generators
  • Accumulation of CO

Our project:

  • Fragrance
  • Energy-independent
  • Transportable
  • Cost-effective
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SLIDE 3

EXPERIMENTATION

CO pathway (2010 METU Turkey) + Wintergreen pathway (2006 MIT) =

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SLIDE 4

T7 CooA

CooA binding site pCooF BSMT

Part Description CooA CO transcription activator T7 T7 promoter pCooF Promoter activated by CO-CooA pchBA pchA: isochorismate synthase pchB: isochorismate pyruvate-lyase BSMT SAM benzoic acid/salicylic acid carboxyl methyltransferase I Chorismate Endogenous molecule Methyl salicylate Wintergreen smell molecule CO pathway Wintergreen pathway

PchA and PchB

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SLIDE 5

CHEMICAL PATHWAY

Chorismate Isochorismate Salicylic Acid Methyl Salicylate

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SLIDE 6

DESCRIPTION

CO pathway

CooA transcription activator T7 promoter

Wintergreen pathway

pchBA and BSMT genes pCooF promoter

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SLIDE 7

CONSTR UCTS

  • E. coli OneShot TOP10

Vectors: pCR2.1-TOPO-TA, pCDFDuet-1 T7-GFP (pCDF) pCooF-GFP (pCDF) T7-CooA (pCR 2.1)

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SLIDE 8

CO EXPERIMENT

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SLIDE 9

EXPERIMENTAL CONSTRUCTS

BL21(DE3) Experiment

T7-CooA, pCooF-GFP

Controls

Negative: DE3 cells only Negative: pCooF-GFP Positive: T7-GFP

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SLIDE 10

CORM-2

  • Carbon monoxide releasing molecules
  • CO in solution
  • Safety
  • Various concentrations
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SLIDE 11
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SLIDE 12

PRELIMINARY METHODOLOGY

1) Anaerobic chamber 2) Cultures to 0.6 OD600 w/ glucose 3) IPTG induction 4) Add CORM-2 (100 µM) 5) Grow anaerobically 6) Fluorescence Intensity

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SLIDE 13

no CORM CORM Series1 69675.53181 61376.63119 56000 58000 60000 62000 64000 66000 68000 70000 72000

Fluorescence

Fluorescence vs. CO presence

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SLIDE 14

MODIFICATIONS

  • Induced for 20 hours
  • Springs to increase aeration
  • Aerobic incubation
  • Grow to 0.6 OD600 in flasks
  • 15mL conicals
  • Inducing at 37°C
  • Multiple CORM concentrations
  • Triplicates
  • Resuspending in PBS
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SLIDE 15

20000 40000 60000 80000 100000 120000 140000 160000 180000 100 200 300

Fluorescence

CORM concentration (uM)

Fluorescence vs. CORM concentration in LB/PBS

LB PBS

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SLIDE 16

FINAL METHODOLOGY

1) Anaerobic chamber 2) Cultures to 0.6 OD600 with glucose 3) IPTG induction 4) Add CORM-2 5) Grow anaerobically 6) Aerobically 7) Resuspended PBS 8) Fluorescence Intensity

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SLIDE 17

DATA

10000 20000 30000 40000 50000 60000 70000 50 100 200 500

Fluorescence CO-RM (uM)

Normalized Fluorescence v. CORM concentration under IPTG induction

DE3 pCoof-GFP T7-CooA pCoof-GFP T7

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SLIDE 18

DATA WITHOUT T7-GFP

100 200 300 400 500 600 700 50 100 200 500

Fluorescence CORM (uM)

Normalized Fluorescence v. CORM concentration under IPTG induction, no positive control

DE3 pCoof-GFP T7-CooA pCoof-GFP

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SLIDE 19

DISCUSSION

Time constraint – CO construct tested only Possible errors

CORM Anaerobic chamber DO content Mutation between transforming plasmids

Obstacles

Sequence verification Creating anaerobic environment Designing primers

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SLIDE 20

FUTURE PLANS

  • Biosensor
  • Anaerobic media
  • Detecting other gases
  • Other smells
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SLIDE 21

SAFETY

  • Lab training
  • Chemicals

CORM-2 DMSO EtBr

  • Fume hood during experimentation
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SLIDE 22

EDUCATION & PUBLIC ENGAGEMENT

KUT KXAN

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SLIDE 23

EDUCATION & PUBLIC ENGAGEMENT

Synthetic Biology Club 2015 SXSW Create

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SLIDE 24

ACKNOWLEDGEMENTS

  • Dr. Ellington and all the members of the Ellington Lab
  • Dr. Mishler and the UT iGEM team
  • Dr. Davies

Chris Cervini Joseph Oleniczak

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SLIDE 25

SPONSORS