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Introduction

Modify E.Coli by adding LuxCDABE geneso that, it can detect mutation.

E. Coli will able to emit blue-green lightwhen placed inserted medium containingpollutant such as methanol and phenol.

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Pollutants detected (Van DYK et

al., 1994)

Ethanol, methanol

2,4-dinitrophenolPhenol

2,4-Dichlorophenoxyacetic acid

pentachlorophenol

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Alternative gene

Promoter is grpE (encode proteininteract with dnaK and groEL) (VanDYK et al., 1994)

Reporter gene is luxCDABE from V.fischeri

Enhance hydrophobic compounddetection by inducing mutation ontolC (Van DYK et al., 1994)

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Gene construct (Bluth, Frew &McNally, n.d.)

dnaKp will act as the promoter

Lux CDABE will act as the reporter where it has

the luminescence ability Rho-independent terminator will terminate the

sequence once it is done expressing.

dnaKp lux CDABERho-

independentterminator

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dnaKpromoter (dnaKp) Isolated from lambda phage 9E1 (Kohara collection) (Van DYK et al., 1994) 192bp Is activated when cell exposes to pollutants

dnaK dnaKencodes for Hsp70 (Van DYK et al., 1994) Expressed when the cell is facing stress (Van DYK et al., 1994) such as

Heat Viral infection Presence of abnormal protein Ethanol 2,4-dinitrophenol

Sodium azide Hydrogen peroxide Heavy metal

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Reporter gene- lux CDABE

Isolated from V. fischeri (Van DYK et al., 1994)

Used for biolumiscence lux CDE is gene for fatty acid reductase enzyme complex

(transferase, synthetase and reductase) (Bluth, Frew & McNally,n.d.)

Conversion of fatty acids into long chain aldehyde

lux Aand lux Bencode for a and b subunit of luciferase (Bluth,Frew & McNally, n.d.)

FMNH2+ RCHO + O2----> FMN + RCOOH + H2O + light (490nm)

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Luciferase

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Rho-independent terminator a G+C rich hairpin loop, followed by a poly-T stretch

of DNA (Bluth, Frew & McNally, n.d.)

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COMPONENTS Origin of replication Responsible for the replication of vector in the

bacterial cell

Tetracycline resistant gene () Selection of recombinant transformant

PromoterPtet Express tetracycline resistant gene

VectorpUCD615 Contains all the gene components Except promoter for lux CDABE (Van DYK et al.,

1994)

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Vector

This is the vector withoutdnaK promoter.

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CONSTRUCTION OF pRY002

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5-GTTAGCGGATCCAAAAGCACAAAAAAT-3

3-ACCTCTGCAAATCTACCTTAAGTGACGA-5

PCR

5-GTTAGCGGATCCAAAAGCACAAAAAAT-3

3-ACCTCTGCAAATCTACCTTAAGTGACGA-5

BamHI

EcoRI

EcoRI sticky end

BamHI sticky end

dnaKp(192bp)

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pUCD615 does not contain any promoter. Inorder to place in the promoter. First,restriction enzyme (BamH1 and EcoRI) areused to cleaved the plasmid. Then thepromoter is inserted. It can be easily inserted

because it contains sticky ends.

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Final vector

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2transformation

E. coli

pRY002

Rho-independentterminator

lux CDABE

ori

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Detection method

Grow to early log phase at 25in LB medium(GIBCO BRL) with 50ug of tetracycline per ml

Measure turbidity with Klett-Summerson

colorimeter When Klett reading is 15 to 30, it is used for stress

induction (Van DYK et al., 1994)

Add pollutants to LB-tetracyclin medium

Measure bioluminescence (at below

30)Dynatech ML3000 microtiter plateluminometer (Van DYK et al., 1994)

Rate of light increase(Relative light unit perminute)= change in RLU per minutes of interval

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Reference Bluth, B.J., Frew, S.E. & McNally, B. (n.d.). Cell-

cell communication and the luxoperon in Vibriofischeri. Retrieved from

https://www.bio.cmu.edu/courses/03441/TermPapers/97TermPapers/lux/default.html Van DYK, T.K., Majarian, W.R., Konstantinov, K.B.,

Young, R.M., Dhurjati, P.S. & Larossa, R.A. (1994).Rapid and sensitive pollutant detection byinduction of heat shock gene-bioluminescence

gene fusions.Applied and EnvironmentalMicrobiology, 60(5), 1414-1420. doi:0099-2240/94/$04.00+0