Imperial Journal of Interdisciplinary Research (IJIR)

Vol-3, Issue-1, 2017

ISSN: 2454-1362, http://www.onlinejournal.in

Construction of Modified pTRG Vector as

pTRGQQ used in Bacterial Two Hybrid Assay for
Protein-Protein Interaction Studies of Dengue Virus
Type-2 Protease with Peptide Libraries
Sushil Kumar1,2,3* & Seema Dwivedi2,
1

Special Centre for Molecular Medicine,

Jawaharlal Nehru University, New Delhi-110067, India
2
School of Biotechnology, Gautam Buddha University, Greater Noida-201308, India
3
Recombinant Gene Product group, International Centre for Genetic Engineering and
Biotechnology, Aruna Asaf Ali Marg New Delhi-110067, India
Abstract: In molecular biology for cloning
experiments, plasmid, a circular molecule is used
as vector (carrier) to insert a desired gene or DNA
fragment by ligating into it after digestion of both
with the compatible restriction enzymes. Then by
transforming with a host (generally E. coli) strain
of desired traits the screening for appropriate
clone is done. A plasmid is having selectable
marker in the form of antibiotic resistant gene
which helps a molecular biologist for the proper
selection of desired clone while screening is done.
Apart from having drug (antibiotic) resistance gene
a plasmid also have an origin of replication and
restriction sites where a gene or DNA can be
inserted
without
interfering
with
plasmid replication or expression of the drugresistance gene. For protein- protein interaction
studies, yeast two-hybrid screening is traditionally
used. It is however a tedious procedure, limited by
the basic biology of the yeast. Yeast grows slowly,
is difficult to transform efficiently and requires
unique reagents and techniques. The bacterial twohybrid system moves two-hybrid studies from yeast
into E. coli a much more convenient host with a
fast growth rate, high transformation efficiency and
easy manipulations. To carryout bacterial two
hybrid assay, two plasmids are required, one which
carries a gene of interest bait (pBT), and another
having a target gene (pTRG). Library which is a
collection of multiple DNA fragments is screened
for desired clones based on blue and white color
appearance on X-gal indicator plate. In this article
we report construction of a new modified plasmid
vector pTRGQQ derived from pTRG vector which
is highly compatible for blunt end cloning at SnaB1
restriction site of target gene into it to carry out
bacterial two hybrid assay.

Imperial Journal of Interdisciplinary Research (IJIR)

1. Introduction
Host and pathogens cross-talk through proteins,
metabolites, small molecules, and nucleic acids,
such as non-coding RNAs [1]. Proteinprotein
interactions (PPI) play a central role in biological
systems [24] defining the interactome of an
organism, or, as elegantly expressed by Robinson
et al. the molecular sociology of the cell [5]. Many
experimental approaches are used to investigate
PPI, including yeast two-hybrid, [6, 7] tandem
affinity purification [8, 9] and mass spectroscopy
[10]. Bacterial two hybrid assay to study proteinprotein interaction has been emerged as a
promising strategy in recent years, as the screening
is faster and convenient in comparison with yeast
two-hybrid assays. Here we report, construction of
a modified bacterial two hybrid vector pTRGQQ,
to enhance the cloning efficiency for proteinprotein interaction studies.

2. Materials and methods

2.1 Construction of pTRGQQ plasmid
644 base pairs (bp) HBHA gene containing
plasmid pUC57 was grown overnight in 100 ml
Luria Bertani Broth (LB) supplemented with
100g/ml ampicillin at 37C. Preparation of DNA
was followed by digesting HBHA gene fragment
with Not1/Xho1 restriction enzymes. This 644 bp
DNA fragment was cloned in Not1/Xho1 enzymes
cut original pTRG vector which resulted in the
formation of HBHA pTRG plasmid (Fig. 1). The
newly constructed HBHA pTRG plasmid was
further digested with SnaB1 restriction enzyme and
allowed to self ligate in an overnight ligation
reaction which resulted in the formation of
pTRGQQ plasmid (Fig. 2) QQ was an addition to
the name of SnaB1 digested self ligated HBHA
pTRG plasmid to differentiate from other plasmids

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Imperial Journal of Interdisciplinary Research (IJIR)

Vol-3, Issue-1, 2017
ISSN: 2454-1362, http://www.onlinejournal.in
(pTRG and pTRGnn). PCR was performed and
sequencing of pTRGQQ plasmid was done to
authenticate the cloning.

2.2 Cloning of genes in two hybrid vectors

To check the cloning efficiency of the
pTRGQQ as two hybrid vector, Mycobacterial
proteins CFP10 and ESAT6, that are known to
form a tight 1:1 complex [11] and have been shown
previously to interact using a bacterial two-hybrid
system [12], were PCR amplified and digested with
SnaB1 restriction enzyme. These genes were
cloned into SnaB1 digested pBTnn and pTRGQQ,
respectively, and used as positive controls for two
hybrid assay. Dengue Virus (DV) type-2 protease
NS3:2b gene was PCR amplified and the 751 bp
PCR product was digested with SnaB1 restriction
enzyme and cloned into SnaB1 digested pBTnn
vector while Human cDNA lung library was
already cloned in pTRG vector. NS3:2bpBTnn and
Human cDNA lung library cloned in pTRG vector
was used as test for bacterial two-hybrid assay.
Empty pBTnn plasmid was used as negative
control with ESAT6 pTRGQQ.

2.3 Bacterial two hybrid assay

E.coli reporter strain R1 was co-transformed
with equal amounts of DNA of positive control
(CFP10pBTnn and ESAT6pTRGQQ), test (NS3:2b
pBTnn and human cDNA lung library cloned in
pTRG) and negative (pBTnn and ESAT6pTRGQQ)
controls separately. The co-transformants were
plated on X-Gal indicator plates containing
kanamycin (50 mg/ml), chloramphenicol (30
mg/ml), tetracycline (12.5 mg/ml), X-Gal (80
mg/ml), Isopropyl b-D-1-thiogalactopyranoside (25
mM)
and
phenylethyl-b-D-thiogalactoside
(200mM). Appearance of blue color colonies on XGal indicator plate was the confirmation of
interaction between proteins CFP10 and ESAT6 as
positive control and interaction with dengue
NS3:2b protease with human cDNA lung library as
test was also confirmed (Fig. 3A) For further
verification of the interaction, positive control
(CFP10pBTnn and ESAT6pTRGQQ) and test
plasmid (NS3:2bpBTnn and its unknown

Imperial Journal of Interdisciplinary Research (IJIR)

interacting partner from human cDNA lung library

in pTRG vector)
were separated by using
chloramphenicol (30 mg/ml) and tetracycline (12.5
mg/ml) containing LB agar plates separately. The
single isolated colony appeared at these plates were
picked and re-grown overnight at 37C in
chloramphenicol (30 mg/ml) and tetracycline (12.5
mg/ml) antibiotic containing LB media. DNA was
prepared and interaction was confirmed by
repeating co-transformations.

2.4 Liquid -galactosidase Assay

All interactions were further verified by performing
liquid -galactosidase assay. Individual colonies
from each co-transformants from X-Gal indicator
plate were selected and grown to mid-log phase in
liquid culture in the presence of 10 M IPTG and
suitable antibiotics. Optical density (OD600) of each
culture was measured at 600 nm. 10 part culture of
negative control and one part culture of positive
control was used while other (test sample) culture
were taken as half of the positive control culture for
performing the assay. The cell pellet was washed
with 1 ml of Z buffer (60 mM Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, and 1 mM MgSO4; pH 7.4)
and resuspended in 150 l of Z buffer containing
40 mM merceptoethanol (-ME), 50 l of
chloroform, and 20 l of 0.1% SDS. The mixture
was vortexed vigorously . The assay was initiated
by adding 700 l of pre-warmed ONPG (Sigma)
substrate from 1 mg/ml (3M) stock solution
(prepared in Z buffer with -ME). The tubes were
incubated at 30C for appropriate time and the
reaction was quenched with the addition of 0.5 ml
of 1 M Na2CO3. The reaction mixture was
centrifuged at high speed in a table top centrifuge
for 10 minutes at room temperature and absorbance
of the clear supernatant was measured at 420 nm.
Enzyme activity (in Millers unit) was calculated
using the equation; Millers Unit = [1000 x A420]/
[Time (in minutes) x Volume (in ml) x A600] (Fig.
3B)

3. Figures
1) Schematic representation of construction of
pTRGQQ vector

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Imperial Journal of Interdisciplinary Research (IJIR)

Vol-3, Issue-1, 2017
ISSN: 2454-1362, http://www.onlinejournal.in
for/pTRG rev primers, lane 1: 1kb ladder, lane 2:
amplified 644bp HBHA gene. (C) colony PCR of self
ligated pTRGQQ (with pTRG for/pTRG rev primers)
lane 1-3 & 5 showing +ve bands on gel is confirmation
of construction of vector, lane 4; 1kb ladder.

3) Bacterial two hybrid assay & Liquid galactosidase assay

Figure 3. (A) Bacterio-Match two hybrid reporter strain

R1 was co-transformed with individual plasmids

ESAT6pTRGQQ+CFP10pBTnn as positive control;
cDNApTRG (human cDNA lung library+NS3:2bpBTnn)
as test; along with ESAT6pTRGQQ+pBTnn as negative
control and plated on X-gal indicator plate, blue color
colonies were the confirmation of interaction; (B) Liquid
- galactosidase assay further confirms the interactions
of ESAT6 with CFP10 and an unknown binding partner
from human cDNA lung library with NS3:2b dengue
type-2 protease.
Figure 1. pDRAW 32 DNA analysis software was used
to generate above vector maps for the exact
representation of the strategy applied in construction of
the pTRGQQ vector.
2) Agarose gel electrophoresis

Figure 2. Agarose gel electrophoresis of 644 base pairs

HBHA gene cloned in original pTRG vector, (A)
:digestion of HBHA containing PUC57 vector DNA with
Not1/Xho1 restriction enzymes, lane 1&3 digested DNA
showing 644bp fall out of HBHA gene; lane 2: 1kb
ladder. (B) colony PCR of HBHA pTRG with pTRG

Imperial Journal of Interdisciplinary Research (IJIR)

4. Results & discussion

In our efforts to carryout bacterial two hybrid
assay for protein-protein interaction studies of
Dengue virus type-2 protease NS3:2b with human
cDNA lung library, we needed highly efficient two
hybrid vectors for blunt end cloning. Vector pBT
and pTRG to clone gene of interest and target gene
are used for bacterial two hybrid assays with
required modifications. In our case for blunt end
cloning, we have been earlier using vector pBTnn
(with SnaB1 restriction site) which is derived from
pBT vector but we were facing problem with
earlier used pTRGnn vector (with SnaB1 restriction
site) derived from pTRG vector. So, we decided to
further modify pTRG vector for our experimental
suitability to study protein- protein interaction. We
were successfully able to clone and re-clone target
as well as gene of interest in newly modified
pTRGQQ vector at SnaB1 site.

5. Conclusion
In an attempt to improve protein-protein
interaction studies based on bacterial two hybrid
assay, we successfully modified pTRG vector
resulting in new version of it as pTRGQQ for blunt

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Imperial Journal of Interdisciplinary Research (IJIR)

Vol-3, Issue-1, 2017
ISSN: 2454-1362, http://www.onlinejournal.in
end cloning at SnaB1 restriction site. Due to its
tenacity, the cloning efficiency of this vector was
found to be very much improved as we cloned
ESAT6 gene into it, which is known to interact
with CFP10 protein. We were also able to found a
new unknown interacting partner of Dengue virus
type-2 NS3:2b protease from human cDNA lung
library, which has to be further studied. Liquid galactosidase assay results further proved the
successful creation of modified pTRGQQ vector
for molecular cloning used in protein- protein
interaction studies.

Acknowledgements
The authors of this research work would like
to thanks Department
of Biotechnology
Government of India for financial support.

[9] Krogan, NJ., Cagney, G., Yu HY., Zhong, GQ., et.al..

Global landscape of protein complexes in the yeast
Saccharomyces cerevisiae, Nature 2006, 440:637643.
[10] Zhou, M., and Robinson, CV., When proteomics
meets structural biology, Trend Biochem Sci 2010,
35:522529.
[11] Renshaw, P. S., et al. Structure and function of the
complex formed by the tuberculosis virulence factors
CFP-10 and ESAT-6, EMBO J. 2005, 24, 24912498.
[12] Tharad, M., et al. A three-hybrid system to probe
in vivo protein-protein interactions: application to the
essential proteins of the RD1 complex of M.
tuberculosis, PLoS ONE 2011, 6, e27503.

Competing interests
No competing interest.

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