E-CO Sensor with Enhanced Dynamic Range (EDR)

Why CO?  Odorless and tasteless gas > Difficult to sense  Extremely toxic A serious threat to human life (Carboxyhemoglobin formation)  A bioplatform to detect a toxic gas using synthetic biology approach  Biosensing of CO is a new area > Understanding molecular basis

 CO as sole carbon source Coo Regulon

 pCooF [pF] and pCooM [pM] promoters  Transcriptional activator: CooA [COA]

• Heme containing gas sensor protein • Belongs to cAMP receptor protein (CRP) and fumarate nitrate reduction (FNR) family of transcriptional regulators • Homodimer • Dimerization ~ Specificity • Response Elements (Mutation target sites) • Heme group (N-terminal) • CO binding > COA activation

Objectives  To construct a carbon monoxide biosensor  To increase the dynamic range of the sensor via strong/weak promoter coupling; Enhanced Dynamic Range (EDR)

Execution - Design and synthesize promoter and CooA variants - Overexpress and purify CooA - Amplify Response Elements - Study promoter-CooA binding affinity - Couple strong/weak promoters with cloning - Conduct cell sensor experiments

Thorsteinsson at al (2000)

Confirmation by Yellow Color

NC pTriEx-CooA

CooA 49 kDa

SDS-PAGE of crude lysate

 One of the biggest challenges

SDS-PAGE of fractions from chealating sepharose column

Expression  Vector-host combinations  IPTG concentration  Post-induction time  Ferric citrate effect  Soluble vs insoluble fraction recovery Purification  Strong anion exchange chr.  Chealating column

Confirmation by Soret Peak

• Elucidation of DNA-Protein interaction • Electrophoretic mobility of cooA – promoter (pcooF and pcooM) complex less than free promoter sequences

Conformation change in the tryptophan residues on cooA cause the decrease in the intensity of fluorescence pink:reduced - CO bounded CooA + buffer yellow : reduced –CO bounded CooA + 28 nM pCooF ( 20. Min) blue: reduced –CO bounded CooA +56 nM pCooF ( 40. Min) green: NC: reduced –CO bounded CooA + 10 ul elution buffer ( 20.min) purple: NC: reduced – CO bounded CooA+ 20 ul elution buffer (40. Min )

Culturing and CO Feeding Setups Fermentor

Flask

Sparging

Flushing

Liquid Medium Planktonic Growth

Controlled Atmosphere Chamber

Solid Medium Biofilm Growth

pECO-version 1.0

Chasis: E.coli BL21 DE3

Fermentor Study

Fluorescence Spectroscopy

pECO-version 1.0e

More in wiki…

pECO-version 2.0

Fluorescence Spectroscopy data

pCooM-RFP CooA IPTG CO

+ -

+ + -

+ + -

+ + + -

+ + +

+ + + +

Negative Control

( - ) IPTG Mode: CLSM Excitation: 543 nm

( + ) IPTG

 We have built a carbon monoxide sensing device which we call E-CO sensor and showed that it works in E.coli !  17 biobricks have submitted to iGEM library  We successfully completed CooA expression and purification work package  Two binding characterization methods, EMSA and ITF, were optimized for affinity screening of mutated promoters  We also optimized three alternative culturing and CO feeding setups for testing and performance evaluation of a gas sensing biosensor  We showed by two independent experiments (EMSA and Cell sensor) that COA has a low-level transcriptional activity even in the absence of CO

 Sensitivity and response time studies  Implementation of Enhanced Dynamic Range (EDR)  Thermodynamic characterization of response element-COA binding via Isothermal Titration Calorimetry (ITC)  Construction and performance evaluation of the new generation E-CO sensors

TERRAFORMING

BIOHYDROGEN PRODUCTION

• Cell population vs fluorescence intensity (Quenching effect) • BBa_J04450 (RFP coding device) • Zhang et. al. (2010) • Model applicability may depend on construct and/or host organism

BIO GUIDE by METU-TURKEY-SOFTWARE Team

SURVEYS METU-TURKEY SOFTWARE Team’s Survey on the development of a new registry interface Mexico-UNAM-CINVESTAV Team’s Survey on ethics of synthetic biology