1. The study of gene expression in Escherichia coli of 5 constitutive promoters.
Choong. M. Y.Y 1, Aldsworth.T 1 and Naseby.D.C1
1 Microbiology, Molecular Biology and Biotechnology Research Group, School of Life Sciences , University
of Hertfordshire, Hatfield, Al10 9AB, Hertfordshire
Introduction
.
Bacterial genes are regulated as clusters of genes, called an operon which is transcribed as a single
mRNA product. The operon is under the control of a regulatory signal or promoter.
Gene Regulation gives the cell control over structure and function, and is the basis for cellular
differentiation, morphogenesis and the versatility and adaptability of any organism. Gene regulation
may also serve as a substrate for evolutionary change, since control of the timing, location, and
amount of gene expression can have a profound effect on the functions of the gene in a cell or in a
multicellular organism. Constitutive gene expression occurs when a gene is transcribed continuously
and is a vital function. However, different constitutive genes are expressed at different levels under the
control of different constitutive promoters. The strength of the promoters are related to the affinity of
RNA polymerases that binds to the promoter sequence. ( Jensen and Hammer, 1997) A greater
understanding of constitutive gene expression could lead to development of better expression systems
for industrial and research purposes.
In this study, the expression by five promoters was studied using a lux reporter system in E coli.
The 5 constitutive promoters are Lys-Lux ; Lysyl-tRNA synthetase, Spc-Lux; Ribosomal protein , Tat –
Lux; Twin arginine Translocation, Ldcc-Lux ; Lysine Decarboxylase and Lpp-Lux; Outer membrane
Lipoprotein.
Lys- lux promoter controls aminoacyl-tRNA synthetase. There are two genes that are regulated, lysS
and lysU. However, lysS is expressed under all growth condition where as lysU is only express under
selective conditions.
The Spc operon controls the regulation of most ribosomal proteins and RNA. The function of spc
operon is shared by str operon with similar sequences.
The TatABCE operon functions as protein translocation pathway. Specific targeting and transport
mechanisms are therefore required to move these proteins from their site of synthesis in the
cytoplasm to their final subcellular destination.
The Lpp promoter controls the expression of the major outer membrane lipoprotein, the
most abundant protein in E.coli. Outer membrane lipoprotein expression is crucial to maintain the
permeability of the cell and formation of the outer membrane vesicles
The fifth constitutive promoter controls expression of lysine decarboxylase (ldc). Basic amino acids
such as lysine, arginine and ornithine are significant physiologically to Escherichia coli. There are a
few transport systems that mediate the basic amino acids pathways.
This study aims to assess the relative expression levels of the five constitutive promoters to gain a
greater understanding of relative promoter strength
Methods
All E.coli strains contained the appropriate promoter fused to the Photorhapdus lux CDABE cassette on the
expression plasmid pBR322 which also contains ampicillin resistance gene. E.coli ATCC 8739-322- Lys-
Lux, E. coli ; E.coli ATCC 8739-322- Spc-Lux ; , E.coli ATCC 8739-322- Tat-Lux ; Twin-Arginine
translocation , E.coli ATCC 8739-322- Ldcc – Lux ; E.coli ATCC 8739-322- Lpp-Lux ( University of
Hertfordshire) ,
Overnight culture
A loop of bacteria from each plate from the 5 different promoters are mixed and vortexed into 15.0 ml of
nutrient broth containing 100ppm ampicillin and grown overnight at 32°C.
Preparation of assay
•50µL of the overnight culture was added into 70mL to Nutrient broth containing 100ppm of ampicillin. This
was incubated in a shaking incubator at 32 °C and 100Rev/min for 24 Hours.
•2.0 ml of the culture was added into 18ml of Buffered Peptone water, further serial dilutions were carried
out from 10-2 to 10 -8 . Triplicate 3.0 ml samples of each dilution was dispensed into a culture tube loaded
loaded into a Celcius Advance luminometer to measure the Relative Light Units ( RLU). Duplicate 1000ul
of each dilution was added to empty petri dishes and 20ml of molten nutrient agar was added.
Results
0
0.2
0.4
0.6
0.8
1
1.2
LPP TAT LDC SPC LYS
Lightpercell
Promoter
0
0.5
1
1.5
2
2.5
3
3.5
4
LPP TAT LDC SPC LYS
LogRelativeLightunits
Figure 1.0: Log Relative Light Units of a 10-3 dilution of
early stationary phase E coli marked with lux CDABE under
the control of 5 Constitutive Promoters
Figure 3.0: The Linear Relationship of Log Relative Light Units (RLU) and Log Colony Forming Units (CFU)
The coefficient of determination, R2: for LPP : 0.9922 , SPC; 0.972 , TAT ; 0.9795 , LYS; 0.9427 SPC ; 0.9601
Results and Discussion•The value of RLU corresponds to the expression of lux . High RLU values therefore correlate to greater promoter strength. As
indicated in Fig 1.0 and Fig 2.0 the greatest lux expression was found under the control of the Lpp promoter, followed by TAT> LDC>
SPC>LYS. The binding affinity of σ factor –RNA polymerase during the initiation process plays an important role in gene expression,
the differences in expression levels of the five promoters investigated are therefore linked to the binding affinity of the polymerase
to the different sequences (Jensen and Hammer, 1997).
•The expression coincides with the importance of different biosynthesis in E. coli . Lpp Promoter is among the strongest, it controls
the expression of outer lipoprotein, the most abundant protein in E coli. The major outer membrane lipoprotein of E.coli is first
synthesized as a precursor protein. The purpose of lipoprotein is to anchor proteins to carry out important functions efficiently at the
membrane – aqueous interface. This also maintains the cell surface integrity by connecting the outer membrane to murein ( Fadi et
al.., 2005).
• The Tat Promoter controls the protein translocation pathway. The tat pathway is capable of exporting prefolded proteins. The tat
expression appears to be the second highest expression after Lpp due to the excess of 20 gene products that are likely to be exported
by the Tat system( Jack et al., 2001). Ldc controls the lysine decarboxylase, Spc on the other hand controls most of the RNA and r
protein synthesis whilst the lowest expression was found with Lys which controls expression of the aminoacyl- tRNA synthetase
which is found in much lower quantities in the cell. All the 5 promoters are expressed constitutively, however the rate of expression
differs. This may be useful information in applications where selecting the appropriate level of expression is required.
•Figure 3.0; shows the linear response for all five constitutive promoters against cfu. The coefficient of determination, R2 is the
measure of the degree of variation. The coefficient of determination for all 5 promoters are between 0.96- 1.0 with a linear response
between approximately 10 -4 and 10 -8 depending on the strain used. This means that the points on the regression line give a good
approximation of the data points.
• The reliability of these lux marked E.coli strains as an estimate of the viable population has potential for use as a sensitive and real
time measurement for use as a viable cell biosensor.
Reference
Jensen,P.H and Hammer,K ( 1998). App Environ Microbiol 82:27-
Liu,X ., Germaine,K.,Ryan,D.,and Dowling.,D (2010). Sensors ,10,1377-1398
Jack,R.,Sargent,F.,Berks,B.,Sawers,G and Palmer,T(2001). Journal of Bacteriology,1801-1804
0
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0 1 2 3 4 5 6 7 8 9 10
logRLU
log CFU
SPC
LPP
LYS
TAT
LDCC
Figure 2.0: The Ratio of relative light units to colony forming
units for E coli marked with lux CDABE under the control of
5 Constitutive Promoters