Introduction

Modify E.Coli by adding LuxCDABE gene

so that, it can detect mutation.
E. Coli will able to emit blue-green light
when placed inserted medium containing
pollutant such as methanol and phenol.

Heat shock fusion gene
Based on cellular adaptive
responses (Van DYK et al., 1994)
Promoter is dnaK (encode Hsp70)
(Van DYK et al., 1994)
Reporter gene used is the
luxCDABE from V.fischeri (Van
DYK et al., 1994)
Pollutants detected (Van DYK et
al., 1994)

Ethanol, methanol
2,4-dinitrophenol
Phenol
2,4-Dichlorophenoxyacetic acid
pentachlorophenol
Alternative gene
Promoter is grpE (encode protein
interact with dnaK and groEL) (Van
DYK et al., 1994)
Reporter gene is luxCDABE from V.
fischeri
Enhance hydrophobic compound
detection by inducing mutation on
tolC (Van DYK et al., 1994)
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 CDABE
Rho-
independent
terminator
DESCRIPTIONS OF THE GENE
COMPONENTS
dnaK promoter (dnaKp)
Isolated from lambda phage 9E1 (Kohara collection) (Van DYK et al., 1994)
192bp
Is activated when cell exposes to pollutants

dnaK
dnaK encodes 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

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 A and lux B encode for a and b subunit of luciferase (Bluth,
Frew & McNally, n.d.)
FMNH
2
+ RCHO + O
2
----> FMN + RCOOH + H
2
O + light (490nm)

Luciferase
Rho-independent terminator
a G+C rich hairpin loop, followed by a poly-T stretch
of DNA (Bluth, Frew & McNally, n.d.)
COMPONENTS
Origin of replication
Responsible for the replication of vector in the
bacterial cell
Tetracycline resistant gene ()
Selection of recombinant transformant
Promoter Ptet
Express tetracycline resistant gene
Vector pUCD615
Contains all the gene components
Except promoter for lux CDABE (Van DYK et al.,
1994)

Vector
This is the vector without
dnaK promoter.
CONSTRUCTION OF pRY002
5-GTTAGCGGATCCAAAAGCACAAAAAAT-3
3-ACCTCTGCAAATCTACCTTAAGTGACGA-
5
PCR
5-GTTAGCGGATCCAAAAGCACAAAAAAT-3
3-ACCTCTGCAAATCTACCTTAAGTGACGA-
5
BamHI
EcoRI
EcoRI sticky end
BamHI sticky end
dnaKp (192bp)
pUCD615
No
promoter
lux CDABE
BamHI
EcoRI
Cleaved with
BamHI & EcoRI
pUCD615
dnaKp
pRY002

ori
Rho-independent
terminator
pUCD615 does not contain any promoter. In
order to place in the promoter. First,
restriction enzyme (BamH1 and EcoRI) are
used to cleaved the plasmid. Then the
promoter is inserted. It can be easily inserted
because it contains sticky ends.
Final vector

2
transformation
E. coli
pRY002
Rho-independent
terminator
lux CDABE
ori
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 plate
luminometer (Van DYK et al., 1994)
Rate of light increase(Relative light unit per
minute)= change in RLU per minutes of interval
Reference
Bluth, B.J., Frew, S.E. & McNally, B. (n.d.). Cell-
cell communication and the lux operon in Vibrio
fischeri. Retrieved from
https://www.bio.cmu.edu/courses/03441/TermP
apers/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 by
induction of heat shock gene-bioluminescence
gene fusions. Applied and Environmental
Microbiology, 60(5), 1414-1420. doi:0099-
2240/94/$04.00+0