Introduction - 2015.igem.org2015.igem.org/files/poster/LASATX.pdf• Anaerobic chamber (above) •...

Data

Fluorescence was normalized by optical density, subtracting

background fluorescence and absorbance of PBS. Bars indicate

average of samples under identical conditions. Error bars indicate

standard deviation.

IntroductionWe introduced a CO-

sensing mechanism into the

bacteria E. coli by using the

CooA transcription activator

and pCooF promoter to

regulate expression of the

pchBA and BSMT proteins,

converting the endogenous

molecule chorismate into

methy l sa l i c y l a te , t he

wintergreen fragrance, in

t h e p r e s e n c e o f C O .

Problem:

• CO: toxic, colorless,

odorless

• Natural disasters

• Power outages

• Gasoline-fueled devices

to generate power

• CO build-up (CO

poisoning)

• Current detectors are

inaccessible for

blind/deaf

Solution:

• Fragrance-producing

bacterial CO sensor

• Energy-independent

• Transportable

• Cost-effective

• Practical in power

outages/natural

disasters where no

power is available

• Sensory-based:

accessible to disabled

.

MethodologyPreparation

• Gibson assembly: T7-GFP in pCDF, pCooF-GFP in pCDF, and T7-CooA in pCR2.1

• Vectors: pCR2.1-TOPO TA cloning vector (AmpR) and pCDFDuet-1 (SpecR)

• Sequence verification in OneShot TOP10 cells

• E. coli BL21 (DE3) cells were transformed with constructs

• Anaerobic chamber: radiation waste container, inlet/outlet adaptors drill-taped, Vaseline seal

Experiment

• Cultures grown in LB to OD600 with 20% glucose

• Resuspended with IPTG

• CORM-2 added at 0, 50, 100, 200 and 500 uM

• Induced for 2 hours under anaerobic conditions

• Cells washed/resuspended in PBS (phosphate-buffered saline)

• Measure fluorescence intensity (600 nm, 25 flashes)

Constructs

• Experiment = T7-CooA, pCoof-gfp

• Negative control 1 = DE3 cells only

• Negative control 2 = pCoof-gfp (no transcription factor - shows leakage of pCoof promoter)

• Positive control = T7-gfp

AuthorsIsaree Pitaktong, Dylan Danzeiser, Kisha Patel, Pia Sen, Sam Gunn, James

Choi, Ella Pettichord, Sally Jung, Oliver Zhao, Anna Savelyeva, Jasmine

Stone, Caleb Ellington, Anusha Vavilikolanu, Ujwal Punyamurtula, Sofia

Valdivieso, Travis Colbert

Conclusions / Future Plans• CO detection construct itself did not fully function

• Wintergreen circuit and full construct was not tested

• Errors may have influenced our experimental results

For the future:

• Anaerobic testing chamber with minimal oxygen content (prevent leakage: manufactured

chamber, using rubber seal)

• Bacteria plated on blood agar plates, minimizing oxygen content

• More checkpoints – verify sensor functionality, increasing project efficiency / troubleshooting

• Recording data and events more in a timely manner (electronic lab books) – team up-to-date

• Repeat CORM dosage curve – confirm optimal amount CORM in bacterial culture (better

readings/analysis of data)

Possible Errors• Failure of CORM (Carbon Monoxide Releasing

Molecule) to produce significant amount of CO

• Detector failing to express

• Detector failing to function

• Anaerobic chamber not fully flushed (oxygen

remaining, dissolved oxygen content also affects)

or may have had leakages, preventing it from being

a completely anaerobic chamber

• CORM is cytotoxic in aerobic conditions (unreliable,

possible cytotoxicity decreased transformation

efficiency)

Liberal Arts and Science Academy iGEM 2015

Data Analysis• Slight increase in GFP expression from 0 to 500 uM

CO-RM

• Negative controls show variation insignificant (GFP

expression approx. equal in CO dependent and

independent constructs)

• Anaerobic chamber (above)

• Entire construct in Geneious (right)

Acknowledgments2010 METU Turkey Team for stemming the use of the carbon monoxide pathway;

2006 MIT team for stemming the use of the wintergreen pathway.

Dr. Andrew Ellington, Dr. Dennis Mishler and all the members of the Ellington Lab at the

University of Texas at Austin for guiding us through the design and experimental process.