A BPA Biosensor A BPA Biosensor The Bisphenolics : Jason Gardiner, Saul Godkewitsch, Benny Hung, Christopher Kelly, David Lancaster, Anthony Lott, Justin Pahara, Kelly Robinson, Tom Tran, Winnie Xu, Saima Zamir Our Advisors: James Maclagan, Michael Deyholos, Doug Ridgway, Michael Ellison
Edmonton, Alberta, Canada Edmonton, Alberta, Canada UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Edmonton, Alberta, Canada Edmonton, Alberta, Canada UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
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Overview Overview iGEM 2007 iGEM 2007 Binary vector Binary vector BPA Background BPA Background Design Design Results Results Summary Summary Acknowledgements Acknowledgements UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
iGEM 2007 iGEM 2007 Operon was completed with 5 different genes synthesizing butanol. To optimize butanol production the operon requires three additional inserts: 1. ETFA : electron-transfer-flavoprotein α 2. ETFB : electron-transfer-flavoprotein, β 3. Thiolase Protein gel of butanol producing pathway proteins UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Plant Binary Vector iGEM: focus on prokaryotic Gardiner/Deyholos MCS NOS Poly A Tail and simple eukaryotic Left border Right border T-DNA repeat systems T-DNA repeat Extend scope to include Kanamycin plants Resistance pVS1 STA • pCambia vector backbone BiobrickVector 6874 bp • Kan and Chpc selective resistance • Reworked MCS includes BioBrick pBR322 or1 prefix and suffix pVR322 bom de • Easy assembly pVS1-REP UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Bisphenolics Plastic Project Bisphenolics Plastic Project Bisphenol A detecting and degrading bacteria UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Plastics and Pollutants Plastics and Pollutants Plastics : Organic polymers of high molecular weight based on synthetic materials. UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Plastics and Pollutants Plastics and Pollutants UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
The Great Pacific Garbage Patch The Great Pacific Garbage Patch UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
I wouldn’ ’t drink from that t drink from that… … I wouldn UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Bisphenol A (BPA) Bisphenol A (BPA) Bisphenol Bisphenol A A Estrogen Estrogen UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Health Hazards Health Hazards Breast Cancer Prostate Cancer Reproductive Health Adipocytes Homeostasis and Development UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Objective Objective To detect and degrade bisphenol A DETECT DESTROY UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Estrogen Receptor (ER) Estrogen Receptor (ER) BPA stimulates transcription of estrogen–regulated genes We can use estrogen receptor (ER) to detect BPA UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Estrogen Receptor (ER) Estrogen Receptor (ER) • Estrogen receptors are intracellular Dimer of Estrogen Receptor α transcription factors • Two forms of estrogen receptor: ER α and ER β – different affinities • Interested in DNA binding domain and ligand binding domain Ligand Binding Upon Dimerization A/B A/B C C D D E E F F DNA Binding UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
ER α α - -1 and ER 1 and ER α α - -2 Toxicity 2 Toxicity ER Mueller-Fahrnow and Egner (2008) report the toxicity of the ER α DNA binding domain ER α -1 : ER α -2 : Ligand binding domain DNA binding domain To test ER toxicity of in our system we expressed the DNA and ligand domains in E. coli and compared their growth rates over time. UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
ER- -DNA and DNA and Ligand Ligand binding ER binding toxicity toxicity Average growth rate of cultures induced with arabinose Empty vector Ligand DNA UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Bacteria are NOT mammals Bacteria are NOT mammals Escherichia coli Megaptera novaeangliae • Transcriptional regulation differs in prokaryotic E. coli compared to eukaryotic mammalian cells • Thus, to repress transcription we devised a method based upon steric hindrance UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
TetR NOT Gate TetR ER No BPA monomer HO OH BisdB ER Dimer BisdA RFP +BPA= UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Proof of TetR Principle We chose RFP (Red Fluorescent Protein) as a downstream reporter in E.coli because it gives rise to visible, red colonies RFP with no TetR RFP with TetR UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
How do we degrade BPA? UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
BisdA and BisdB Degrade BPA Sasaki et al. isolated two genes that can degrade BPA together Sasaki et al . 2008 UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
BisdA and BisdB Degrade BPA 115ug/ml-90ug/ml over 18 hours of incubation Empty Vector BisdB BisdA/B Sasaki et al . 2008 UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Summary: BPA Absent Without BPA or any estrogen-like compound, ER receptor is made in the cell, as is the TetR transcription factor. TetR 1. ER in the monomer form does not inhibit the production of TetR. ER No BPA ERE is unbound monomer ERE UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Summary: BPA Absent Without BPA or any estrogen-like compound, ER receptor is made in the cell, as is the TetR transcription factor. TetR 2. TetR represses the transcription of RFP, BisdA, and ER No BPA BisdB monomer ERE UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Summary: BPA Present When BPA is present, the ER receptor dimerizes 1. ER dimer binds to ERE, and HO OH represses TetR transcription ER Dimer ER monomer + BPA = ERE UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Summary: BPA Present When BPA is present, the ER receptor dimerizes 2.This allows the upregulation of target genes RFP, BisdA, and BisdB HO OH ER Dimer BisdB RFP BisdA ER monomer + BPA = ere UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Summary: 3 Systems System 1 (Estrogen Receptor): We have the two domains assembled and have confirmed that they are not toxic to the cell. System 2 (TetR): Assembled with the entire operon excluding ER and has been shown to successfully turn off TetR sensitive promotors. System 3 (RFP & BisdA/B): Assembled with the entire operon excluding ER and regulated by TetR. BisdA/B can degrade BPA. Optimization is required. UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
BPA and your research! UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Acknowledgements Acknowledgements Our Sponsors Our Sponsors Our Former Teammates Teammates Our Former Our Consultants Our Consultants UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Overview TetR ER No BPA monomer HO OH BisdB ER Dimer RFP BisdA +BPA= UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
Degradation Products BPA is degraded into two metabolites BisdA & BisdB BisdB Empty vector No cells Sasaki et al . 2008 UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA
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