A Naphthenic Acid Biosensor for Tailings Pond Reclamation - - PowerPoint PPT Presentation

A Naphthenic Acid Biosensor for Tailings Pond Reclamation

University of Calgary iGEM 2011

Water Froth Sand Crusher

Slurry mixture

+ hot water + chemical agents (NaOH) Tailings ponds Open pit mining Separation Extraction plant

Oil Sands Extraction

Gieg L. (2011).

• Recalcitrant carboxylic acids
• CnH2n+zO2

Naphthenic Acids (NAs)

Adapted from Whitby C. (2010).

NAs in tailings ponds are highly toxic to a variety of organisms

You’re Killing Me!

50 50 75 2 11 14 10 20 30 40 50 60 70 80 LC50 (mg/L) 96-hour 60-day

Rutulis frisii kutum (adult kutum) Acipenser gueldenstaedi (sturgeon) Rutulis rutulis caspicus (roach) Compiled from Dokholyan VK., Magomedov AK. (1983).

Social Implications of NAs

Concern about Oil Sands Toxins

Government response

• f increasingly strict

legislation Assessment technologies need to mature alongside process technologies to meet government standards

Stricter Legislation Improved Detection Technologies

Increasing social concern about industrial impact on the environment

What Already Exists?

Roesler R. (2007). and ASU Knowledge Enterprise Development

Gas Chromatography-Mass Spectrometry (GC-MS) Fourier Transform Infrared Spectroscopy (FTIR)

Introducing…

Photos courtesy of Dennis Kunkel and the University of Tsukuba

Pseudomonas putida Pseudomonas fluorescens Dunaliella tertiolecta

The Vision

Tailings In

Response Biosensing Organism Quantitative Output

Building a Biosensor

Naphthenic Acid Detector Reporter Chassis

The Search For a Promoter

Candidate Gene Approach Biotinylation/Immunoprecipitation “Sensory Element Fishing”

Biotinylation

Cyclohexane Pentanoic Acid Biotin Biotinylated Cyclohexane Pentanoic Acid

Biotinylation

Streptavidin Bead

Precipitation

Precipitation

Confirming Biotinylation

Confirming Biotinylation

Successful Biotinylation of Three Naphthenic Acids!

Pull-Down

Candidate Gene Approach

The Search For a Promoter

Biotinylation/Immunoprecipitation “Sensory Element Fishing”

Cooperative Degradation of NAs

Del Rio et al. (2006).

Which Horse Should I Pick?

qRT-PCR Data

Putative Enoyl-CoA Hydratase (Pseudomonas fluorescens LD2)

NA exposure regulates expression of fad-2/Enoyl CoA Hydratase

Up and Down…

GFP RFP

• Designed a novel

protocol to identify small hydrophobic molecule interactors

Current Progress

• Identified a possible

NA-responsive gene

Building a Biosensor: Reporter

lacZ

The CPR Pathway

chlorophenol red-β-D- galactopyranoside chlorophenol red galactose β-GAL

β-Gal Cleaves CPRG

0.5 1 1.5 2 2.5 0:00 0:28 0:57 1:26 1:55 2:24 2:52 3:21 3:50 4:19

Concentration of CPR formed (mM) Time (hrs:min) Concentration of CPR formed

BBa_I732901

IPTG Inducible lacZ

Cyclic Voltammetry

Circuitry and Measurement

Circuitry and Measurement

CPRG Degradation Can Be Detected Electrochemically With Our Construct!

Change in Current (ΔmA) 0 uM 100uM 1mM 0.030 0.039 0.041

Electrochemical Optimization

• Optimized buffering systems
• Cation co-factor inclusion
• Plating technique refined

Nanopure H20 0.1 M CaCl2

Building a Biosensor: Chassis

• E. coli Growth Assay

• New Microalgae Protocols on Our Wiki

– Transformation – Isolation of Chloroplasts – Isolation of DNA

Working with New Organisms

H+ H+ H+ H+ H+

PEG

Agitation Culture

Duneliella is Transformed!

Bright field Gus stain Merge

K

Existing Tools

Selectable Markers

S

35S CaMV

Strong Constitutive Promoter Visual Qualitative Reporter

What Do We Need?

Algae-Optimized Luciferase (BBa_K640000) Heat-Inducible Promoter (BBa_K640001)

HspA70

Bacterial Conjugation

OriT Ori1600 Ori1600

Growth Assay Data

LB Amp Chlor Kan

• E. coli

(Amp Resistant)

• P. putida

LD1

• P. fluorescens

LD2

Conjugation Results

Yellow = Pseudomonas Growth

• E. coli Control

C C C B B

CTRL CTRL

C = Calgary B = Berkeley

Quantitation

(Calgary) (Berkeley)

The Final Construct

Prototype

Circuit Diagram Corresponding Prototype

The Vision

Tailings In

Electrochemical Detector Biosensing Organism Quantitative Output

Market Analysis

Component Price Per Unit ($) Units Cost (CAN $) Prototyping Board 13.70 1 13.70 Box 9.90 1 9.90 Batteries and Connectors 2.64 2 5.28 Electrode Connectors 3.23US 1 3.24 Other Components 1.09US 3 3.27 Wires 18.06US/30.5m 25cm 0.15 Total Cost 35.54 Component Total Cost ($) Units Cost Per Unit ($) LB Broth for Cultures 333.00 125 000 0.0027 Electrodes 200.00 100 2.00 Lab Technician Stipend 25.00/hr 200 0.13 Total Cost 2.13

• Designed a novel protocol to identify small

hydrophobic molecule interactors

• Identified a possible NA-responsive gene
• Characterized a novel reporter function for

the lacZ gene

• Characterized a Pseudomonas-E. coli

conjugation construct

• Submitted parts and protocols for future

work in microalgae and Pseudomonas

• Conducted a market analysis

Accomplishments

Outreach

PATW Bassano Music Video Collaboration Report

• Dr. Arcellana-Panlilio,
• Dr. Nygren, Dr.

Schryvers, Dr. Gieg,

• Dr. Alcantara, Dr.

Muench

• Deirdre Lobb
• Birss Lab
• Cobb Lab
• Dr. Tung
• Erin Brown
• Dr. Moorhead
• Department of Cell

Biology and Anatomy

• Department of

Electrical Engineering

• Department of

Biomedical Engineering

Acknowledgements

Our Sponsors

Collaboration Data

Reaction Mechanism

EDAC

+ +

H2N–R H2N–R

Human Practices

Increasing social concern about industrial impact on the environment Government response of increasingly strict legislation Assessment technologies need to mature alongside process technologies to meet government standards A market for a biosensor that can efficiently detect naphthenic acids in a sample Use of synthetic biology in environmental monitoring paves the road for larger-scale uses in the industry (i.e. remediation) *Please see the U of C team Wiki for more information on current policy and standards*

Circuitry and Measurement

CPRG Degradation Can Be Detected Electrochemically With Our Construct!