A BPA Biosensor A BPA Biosensor The Bisphenolics : Jason Gardiner, - - PowerPoint PPT Presentation

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Sep 19, 2023 335 likes •702 views

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,

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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

A BPA Biosensor A BPA Biosensor

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UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA

Edmonton, Alberta, Canada Edmonton, Alberta, Canada

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UNIVERSITY OF ALBERTA UNIVERSITY OF ALBERTA

Edmonton, Alberta, Canada Edmonton, Alberta, Canada

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Meet eet t the... e...

asds

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Meet eet t the... e...

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Overview Overview

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iGEM iGEM 2007 2007 Binary vector Binary vector BPA Background BPA Background Design Design Results Results Summary Summary Acknowledgements Acknowledgements

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iGEM iGEM 2007 2007

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Protein gel of butanol producing pathway proteins

Operon was completed with 5 different genes synthesizing butanol. To optimize butanol production the operon requires three additional inserts:

1. ETFA : electron-transfer-flavoprotein

2. ETFB : electron-transfer-flavoprotein, β
3. Thiolase

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iGEM: focus on prokaryotic and simple eukaryotic systems Extend scope to include plants

pCambia vector backbone
Kan and Chpc selective resistance
Reworked MCS includes BioBrick

prefix and suffix

Easy assembly

Plant Binary Vector

Gardiner/Deyholos MCS NOS Poly A Tail Right border T-DNA repeat Left border T-DNA repeat Kanamycin Resistance pVS1-REP pVS1 STA

BiobrickVector 6874 bp

pVR322 bom de pBR322 or1

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Bisphenolics Bisphenolics Plastic Project Plastic Project

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Bisphenol A detecting and degrading bacteria

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Plastics and Pollutants Plastics and Pollutants

Plastics: Organic polymers of high molecular weight based on synthetic materials.

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Plastics and Pollutants Plastics and Pollutants

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The Great Pacific Garbage Patch The Great Pacific Garbage Patch

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I wouldn I wouldn’ ’t drink from that t drink from that… …

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Bisphenol Bisphenol A (BPA) A (BPA)

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Bisphenol Bisphenol A A Estrogen Estrogen

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Health Hazards Health Hazards

Adipocytes Reproductive Health Breast Cancer Prostate Cancer Homeostasis and Development

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To detect and degrade bisphenol A

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Objective Objective

DETECT DESTROY

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BPA stimulates transcription of estrogen–regulated genes We can use estrogen receptor (ER) to detect BPA

Estrogen Receptor (ER) Estrogen Receptor (ER)

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A/B A/B C C D D E E F F DNA Binding Ligand Binding Upon Dimerization Dimer

f Estrogen

Receptor α

Estrogen

receptors are intracellular transcription factors

Two forms of estrogen

receptor: ERα and ERβ – different affinities

Interested in DNA binding domain

and ligand binding domain

Estrogen Receptor (ER) Estrogen Receptor (ER)

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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.

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ER ERα α-

1 and ER

1 and ERα α-

2 Toxicity

2 Toxicity

ERα-1: Ligand binding domain ERα-2: DNA binding domain Mueller-Fahrnow and Egner (2008) report the toxicity

f the ERα

DNA binding domain

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ER ER-

DNA and

DNA and Ligand Ligand binding binding toxicity toxicity

Average growth rate of cultures induced with arabinose

Empty vector Ligand DNA

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Transcriptional regulation differs in prokaryotic E. coli

compared to eukaryotic mammalian cells

Thus, to repress transcription we devised a method

based upon steric hindrance

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Bacteria are NOT mammals Bacteria are NOT mammals

Escherichia coli Megaptera novaeangliae

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RFP ER monomer No BPA TetR +BPA= ER Dimer BisdB BisdA

OH HO

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TetR NOT Gate

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RFP with no TetR RFP with TetR

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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

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How do we degrade BPA?

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Sasaki et al. isolated two genes that can degrade BPA together

Sasaki et al. 2008

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BisdA and BisdB Degrade BPA

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115ug/ml-90ug/ml over 18 hours of incubation

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BisdA and BisdB Degrade BPA

Sasaki et al. 2008 Empty Vector BisdB BisdA/B

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ER monomer ERE No BPA TetR

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Summary: BPA Absent

Without BPA or any estrogen-like compound, ER receptor is made in the cell, as is the TetR transcription factor.

1. ER in the monomer form does

not inhibit the production of TetR. ERE is unbound

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ER monomer ERE No BPA TetR

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2. TetR

represses the transcription of RFP, BisdA, and BisdB

Without BPA or any estrogen-like compound, ER receptor is made in the cell, as is the TetR transcription factor.

Summary: BPA Absent

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ERE ER Dimer

When BPA is present, the ER receptor dimerizes

1. ER dimer

binds to ERE, and represses TetR transcription

ER monomer + BPA =

OH HO

Summary: BPA Present

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RFP ere ER Dimer BisdB BisdA ER monomer + BPA =

OH HO

When BPA is present, the ER receptor dimerizes

2.This allows the upregulation

target genes RFP, BisdA, and BisdB

Summary: BPA Present

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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.

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Summary: 3 Systems

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BPA and your research!

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Acknowledgements Acknowledgements Our Former Our Former Teammates Teammates Our Sponsors Our Sponsors Our Consultants Our Consultants

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RFP ER monomer No BPA TetR +BPA= ER Dimer BisdB BisdA

OH HO

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Overview

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BPA is degraded into two metabolites

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Degradation Products

Sasaki et al. 2008 BisdA & BisdB BisdB Empty vector No cells