Enlisting E. Scherichia Holmes Kristina Ehrhardt, Sameer Sant, Jacob - PowerPoint PPT Presentation

Enlisting E. Scherichia Holmes Kristina Ehrhardt, Sameer Sant, Jacob White, Lagnajeet Pradhan, Mitu Bhattatiry University of Texas at Dallas iGEM 2010 Sunday, Nov. 7, 2010 Walker Memorial 54-100

University of Texas Dallas • Established in 1969 with support from T.I. • Located in major tech hub • Fast-growing research institution • Competitive School of Engineering – New Bioengineering Department

Motivation • Deepwater Horizon Spill released 206 million gallons of toxic crude into Gulf of Mexico • Alkanes and Aromatics – Two largest fractions of petroleum by percent composition • Eutrophication: nitrate/nitrite enrichment of water – Algal blooms • Circulate through environment and are resistant to biotransformation

Whole-Cell Biosensors Molecular Input Signal Processor Output Recognition • Visible spectrum reporters (Cambridge 2009) – For cheap, on-site detection of pollutants • E. coli chassis – For well-studied gene expression and widespread use in Registry

Action Plan • Develop whole-cell biosensors to sense trace levels of nitrate/nitrites, aromatics and alkanes – Build new composite BioBricks from existing parts – Characterize existing BioBricks – Build a new BioBrick – Explore combinatorial logic functions

Goals  Nitrate/Nitrite Biosensor  Aromatics Biosensor  Alkane Biosensor  DNA Logic Gate

Nitrate/Nitrite Sensor • Edinburgh 2009 – BioBrick BBa_K216015: pYeaR + LuxAB • University of Texas at Dallas 2010 – Fused pYeaR to GFP and RFP to make new BioBricks – Further expand characterization of pYeaR through titration and time-lapse experiments

Nitrate/Nitrite Sensor

Titration Experiment pYeaR + GFP

Time-lapse Experiment Time-lapse Experiment PyeaR + GFP

Titration Experiments Titration Experiment PyeaR + GFP

Titration Experiment PyeaR + RFP

The Checklist  Nitrate/Nitrite Biosensor  Aromatics Biosensor  Alkane Biosensor  DNA Logic Gate

Aromatics Sensor • Glasgow 2007 – BioBrick BBa_I723032 : pR + XylR • Michigan 2009 – BioBrick BBa_K270003: Pu + GFP (uncharacterized) • UT Dallas 2010 – Characterized BioBrick BBa_K270003: Pu + GFP

Aromatics Sensor Titration Experiment pR + XylR and Pu + GFP

Titration Experiment pR + XylR and Pu + GFP

The Checklist  Nitrate/Nitrite Biosensor  Aromatics Biosensor  Alkane Biosensor  DNA Logic Gate

Alkane Sensor

Progress • Obtained oligonucleotides of PalkB promoter • AlkS gene obtained from Prof. Van der Meer (University of Lausanne) – Planned to PCR the AlkS gene to build a new BioBrick • Performed sticky and blunt-end ligations of PalkB to iGEM backbone.

The Checklist  Nitrate/Nitrite Biosensor  Aromatics Biosensor Alkane Biosensor  DNA Logic Gates

NOR Gate Logic Table Inputs Output Nitrate/Nitrite Aromatic 1 1 0 1 0 0 0 1 0 0 0 1 NAND Gate Logic Table Inputs Output Nitrate/Nitrite Aromatic 1 1 0 1 0 1 0 1 1 0 0 1

Whole-Cell Biosensors Molecular Input Signal Processor Output Recognition • Design signal processing mechanism – Visual reporters (Cambridge 2009) – Sophisticated mechanism for controlling output

BioBricks • Developed new BioBricks – BioBrick BBa_K412000: pYeaR + GFP – BioBrick BBa_K412001: pYeaR + RFP • Characterized new and existing BioBricks – BioBrick BBa_K412000: pYeaR + GFP Expand Edinburgh 2009’s characterization of pYeaR promoter – BioBrick BBa_K412001: pYeaR + RFP – BioBrick BBa_K270003: Pu + GFP (Michigan 2009) • Pursued construction of novel BioBricks – BioBrick BBa_412004: AlkS – BioBrick BBa_412005: PalkB – BioBrick BBa_412006: PalkB + GFP

Acknowledgements • Prof. Leonidas Bleris and Prof. Hyun-Joo Nam • University of Texas at Dallas • Dr. Jan Roelof van der Meer (University of Laussanne) • MIT and iGEM 2010

Thank you!