1. Gene Expression and Preservative Efficacy studies of Five lux-based E.coli
Biosensor strains using novel Bioluminescent Method
Choong.M.Y.Y 1
, Aldsworth,T2
and Naseby.D.C1
1
Microbiology, Molecular Biology and Biotechnology Research Group, School of Life Sciences , University of Hertfordshire, Hatfield, Al10 9AB, Hertfordshire
2
Faculty of Health and Life Science, Coventry University, Priory Street, CV1 5FB, Coventry
.
Introduction
Rapid and sensitive detection of bacteria is very important in the pharmaceuticals, food safety, medical diagnostic, and bio defense. The aim of this study was to validate the gene expression of plasmid bore transcription fusion
between five constitutive E.coli promoters and the Photorhabdus luminescence lux operon as a novel alternative method in enumerating bacteria. The gene expression of these five biosensors was measured using
bioluminescence as relative light units (RLU), and is being validated with three other concurrent methods; traditional plate counting (CFU); ATP-bioluminescent (ATP-B); and fluorescence spectrometry (RLU). These five biosensors
were used to determine the efficacy of Sorbic acid as a preservative in accordance with the European Pharmacopeia regimes. The goal of this research is to replace the time consuming and laborious plate count method with the
bioluminescent approach which is quicker and can be ‘real time’. The five promoters which were used in the study are Outer Membrane lipoprotein (Lpp-E.coli 8739), Twin Arginine Translocase (TatA- E.coli 8739), Lysyl-T-RNA
(LysS- E.coli 8739), Lysine Decarboxylase (LdcC- E.coli 8739) and Ribosomal Protein (Spc- E.coli 8739) (Naseby, 2006).
Materials and methods
 Seven strains of E.coli (5-lux Biosensors, pBR-lux E.coli 8739 and wildtype E.coli ATCC 8739) were grown in 50 ml of TSA with Ampicillin (final concentration of 100μg/ml) at 32°C, 100 Rev/min on an orbital shaker. The growth curves
were constructed based on relative light Units (RLU) measured using Celcius Advance Luminometer through out 48 hours. Spread plate method was carried out for colony forming units (CFU) counts. RLU and CFU were dpne in
triplicates. (n=3).
 ATP measurements were made using ATP-bioluminescence Kit HS II (Roche), whilst ADP and AMP were quantified with addition of enzymes pyruvate kinase, phosphoneolpyruvate (PEP) and adenylate kinase. The adenylate energy
charge (AEC) is calculated based on light readings of ATP, ADP, and AMP.
 AEC = ATP+ 0.5 (ADP) (Chapman et al.,1971)
 (ATP+ADP+AMP)
 Fluorescence spectrometry (RFU) readings were taken using a Promega GloMax detection system with green filters (excitation at 525 nm, emission at 580-640nm) and blue filters (excitation at 490nm, emission at 510-570nm). The
fluorescence dyes were used from Cell viability staining Live/Dead Baclight kit (Molecular Probes).
 10μl of each sample was placed on a microscope slide. Total magnification of 1000x was observed under oil immersion lens fluorescence microscope (Nikon EFD-3) using a mercury lamp (Nikon). Images were taken using a GX CAM
Scientific imaging camera and pictures were edited with GX capture software.
Results
Figures 1.0 & 2.0 : Relationship between bioluminescence and growth phases for each biosensor. Gene expression of lux operon during growth curve shows distinct
phases; lag phase, exponential phase , stationary phase and late stationary phase for 48 hours .The physiological characteristic of the light emission from the five biosensors
resembles significantly (p<0.005) the kinetics of colony forming unit counts with a log difference between the CFU counts and RLU counts.
Biosensor Concentrations
(mM)
Actual
sampling time
to time to≥ 3
log10 CFU
(Days)
% of light
reduction
at 3 ≥ log10
CFU
R2
Values for RLU-
CFU throughout 28
days
Lpp-lux E.coli 35.67 0 99.9 0.647***
17.84 0 99.9 0.563***
8.92 0 99.9 0.563***
4.46 1 99.9 0.595***
2.23 2 99.9 0.496*
1.11 28 99.9 0.510*
0.55 28 99.9 0.765*
0.27 28 99.9 0.754*
Spc- lux E.coli 35.67 0 97.9 0.978*
17.84 0 90.4 0.930*
8.92 0 87.6 0.975*
4.46 7 99.9 0.936*
2.23 7 99.9 0.981*
1.11 28 99.9 0.992*
0.55 28 99.9 0.992*
0.27 28 99.9 0.987*
Tat- lux E.coli 35.67 0 99.5 0.834*
17.84 0 99.9 0.874*
8.92 0 99.9 0.926*
4.46 7 99.8 0.903*
2.23 28 99.0 0.924*
1.11 28 99.4 0.708*
0.55 28 99.9 0.716*
Lys- lux E.coli 35.67 0 96.0 0.840*
17.84 0 96.2 0.772*
8.92 0 99.5 0.773*
4.46 2 99.4 0.700*
2.23 7 99.5 0.750*
1.11 21 99.7 0.888
0.55 28 99.9 0.744*
*
0.27 28 96.6 0.708*
Ldc- lux E.coli 35.67 0 99.9 0.985*
17.84 0 99.9 0.985*
8.92 0 99.8 0.830*
4.46 1 99.9 0.829*
2.23 7 99.9 0.727*
1.11 28 99.9 0.998*
0.55 28 99.9 0.998*
0.27 28 99.9 0.998*
pBr-lux E.coli 35.67 0 N/A N/A
17.84 0 N/A N/A
8.92 0 N/A N/A
4.46 7 N/A N/A
2.23 7 N/A N/A
1.11 28 N/A N/A
0.55 28 N/A N/A
0.27 28 N/A N/A
Table 1.0: Pharmacopeia defined efficacy of sorbic acid within the range of 35.67mM
to 0.28mM at pH 5.0 against five E.coli biosensor and control throughout 28 days
(20°C)
Figures 3.0 & 4.0: Total Adenosine Triphosphate (ATP) measured by enzyme reaction reflects on the metabolic regulation of biosensors. There is an inverse kinetics relation
between the growth phase (Figures 1.0 & 2.0). All biosensors demonstrate similar metabolic activity, as levels of ADP and AMP increase during the long term stationary
phase (336, 504, and 672 hours) and that thus the reporter is not significantly draining ATP from the cells nor it being starved of ATP in a growth dependent manner.
Figure 5.0: Fluorescence spectrometry emission (510-570nm) of live cells at lag phase, exponential and stationary phase. Figure 6.0 and 7.0 represents live
and dead cells of 168 hours observed under the fluorescence microscope using green and red filters. Viability of cell does not decline until 168 days (2 log
units), similar to ATP-bioluminescence.
Discussion and conclusion
Table 1.0 represents the efficacy testing of Sorbic Acid as a preservative.
There is a 99.9% reduction of light which represents a 3 log unit of CFU
reduction. Excellent correlation values (R2: 0.70-0.998) between RLU and
CFU certifies the strong relation between the novel bioluminescent method.
 Validation of the novel bioluminescent method with concurrent methods such as traditional plate counting method, ATP-bioluminescent method and fluorescence
spectrometry gives more confidence and scientific relevance for these five E.coli biosensors to be used in sorbic acid.
 The kinetics of light emission demonstrates that expression of the five promoters are constitutive, and portray distinct growth phases. The Decreasing intensity
of light measured at 24 hours is Lpp> Tat> Ldc> Lys > Spc , with correlation coefficients (R2
) of RLU to CFU as follow; 0.992 , 0.9485 ,0.953 ,0.9418 , and 0.88.
 The limit of detection (LOD) for the biosensors was between 1.16 x 104
to 1.20x 105
CFU, with TatA being the lowest limit followed by LysS, LdcC, Spc and Lpp.
This method offers a good range of sensitivity. The range of sensitivity is the same as ATP-bioluminescence method (104
Cfu/ml) (Gracias and McKillip,2004).
 The bioluminescence method is easy to operate, sensitive, cheap and most importantly, has biological relevance to E.coli. This attribute is not found in chemical
and enzymatic methods of bacteria enumeration.
 The lower correlation between RLU-CFU with sorbic acid preservative suggest, there might be an over expression of Slp, an outer membrane lipoprotein induced
to reduce metabolite stress (Mates, et al.,2007), caused by the disruption of membrane layer under acid stress. Further investigation needs to be carried out to
prove this phenomena.