FIELD OF INVENTION
The present invention relates to a modified vector prepared by ligating a reporter gene
having a STOP codon upstream of the multiple cloning site of the vector whereby the
modified vector when introduced in the host cell does not fluoresce or show color. The
invention also relates to method of producing the modified vector and method of
identification and screening of recombinant clone. It also relates to kit comprising the
said vector.
BACKGROUND OF THE INVENTION
Molecular cloning is an important tool in our endeavor to understand the structure,
function and regulation of individual genes and their products. Molecular cloning
provides a means to exploit the rapid growth of bacterial cells for producing large
amounts of identical DNA fragments, which alone have no capacity to reproduce by
themselves. The power of molecular cloning is remarkable: a liter of bacterial cells
engineered to amplify a single fragment of cloned human DNA can produce about ten
times the amount of a specific DNA segment that could be purified from the total cellular
content of the entire human body.
A recombinant DNA consists of two parts: a vector and the passenger sequence that is
the gene of interest (GOI). Vectors contribute in replication functions due to presence of
origin of replication sequences in the vector. The process of joining the vector and
passenger DNAs is called ligation. T4 DNA ligase enzyme carries out ligation process by
using ATP energy to make the phosphodiester bond between the vector and passenger
sequence. If the vector and passenger DNA fragments are generated by the action of
the same restriction endonuclease, they will join by base-pairing due to the compatibility
of their respective ends. Such a construct is then transformed into prokaryotic cell,
where unlimited copies of the construct and essentially the passenger sequence are
made inside the cell.
Next step is to screen and identify recombinant clones from non-recombinants. There
are numerous methods developed to screen recombinant clones and the methods
include polymerase chain reaction (PCR), blue white screening, vectors carrying lethal

gene which gets inactivated upon insertion of any foreign gene, use of reporter gene
which upon cloning, either fluoresce or the color disappears and other methods in the
art.
The reporter gene which are commonly used are chloramphenicol acetyl transferase
gene (CAT), .beta.-galactosidase gene (.beta.-gal), luciferase gene (luc), alkaline
phosphatase gene (AP), secreted alkaline phosphatase gene (SEAP).beta-
glucuronidase gene (GUS). All fluorescent protein gene (Green fluorescent protein,
Yellow fluorescent protein, Red fluorescent protein, Enhanced green fluorscent protein,
Orange fluorescent protein, Cyan fluorescent protein,) , human growth hormone gene
(hGH) and beta-lactamase gene (.beta.-lac). GFPs from other sources like Renilla and
from Ptilosarcus may also be used. Host cells, including bacterial, yeast and mammalian
host cells, and plasmids for expression of the nucleic acids encoding each luciferase and
GFP and combinations of luciferases and GFPs may also be used in these hosts which
by substitution of codons optimized for expression in selected host cells or hosts, such
as humans and other mammals, or can be mutagenized to alter the emission properties.
On the other hand, various cloning vectors permitting direct selection (positive selection)
of recombinant strains have been described in the scientific literature.
The process of screening bacterial transformants that carry recombinant plasmids
(having gene of interest) is made more rapid and simple by the use of vectors with
visually detectable reporter genes. The blue-white screen is one such molecular
technique that allows for the detection of successful ligations in vector based gene
cloning (Gronenborn and Messing, 1978). The molecular mechanism underlying this
technique is based on genetic engineering of the lac operon present in the laboratory
strain of Escherichia coli that serves as a host combined with a subunit complementation
achieved with the cloning vector.
For example, the vector pBLUEscript, which is commercially available encodes a subunit
of LacZ protein with an internal multiple cloning site (MCS), while the chromosome of the
host strain encodes the remaining O subunit to form a functional beta galactosidase
enzyme which is involved in lactose metabolism. As such, selecting right type of vector
and competent cells are critical considerations to blue white screening. The multiple

cloning site (MCS) can be cleaved by different restriction enzymes so that the foreign
DNA can be inserted within the lacZ gene, thus disrupting the production of functional
beta galactosidase. When E. coli cells containing just the vector are grown in the
presence of an artificial substrate X gal (5-bromo-4-chloro-3-indolyl R galactoside), a
colourless modified galactose sugar that is metabolized by Beta galactosidase, the
colonies turn blue, due to production of active enzyme that gives rise to blue end
product. On the other hand, when the piece of foreign DNA is inserted into the MCS, the
lacZ gene cannot make an active protein fragment and thus functional beta
galactosidase, as a result, colonies of the bacteria that contain cloned foreign DNA
appear whitish. Isopropyl b D-1-thioglactopyranoside (IPTG), which functions as the
inducer of the Lac operon, can be used in some strains to enhance the blue phenotype.
The blue white screen technique involves a screening procedure (discrimination) rather
than a procedure for selecting the clones. Discrimination is based on identifying the
recombinant within the population of clones on the basis of a color. The LacZ gene, in
the vector used for generating recombinants, may be non-functional and cannot produce
beta-galactosidase. As a result, these cells cannot convert X-gal to the blue substance
so the white colonies seen on the plate may not be recombinants but just the
background vector and thus give false results. Moreover, this complex procedure
requires the use of the substrate X-gal which is very expensive, unstable and is
cumbersome to use.
Although the lacZ and many other systems have been extensively used for Gram
negative bacteria like E.coli, there are limited options available for screening
recombinants transformed in Gram positive bacteria.
Chaffin and Rubens in 1998 have developed a Gram-positive cloning vector pJS3, that
utilizes the interruption of an alkaline phosphatase gene, phoZ, to identify recombinant
plasmids. A multiple cloning site (MCS) was inserted distal to the region coding for the
putative signal peptide of phoZ. Alkaline phosphatase expressed from the derivative
phoZ gene (phoZMCS) retained activity similar to that of the native protein and cells
displayed a blue colonial phenotype on agar containing 5-bromo-4-chloro-3-indolyl
phosphate (X-p). Introduction of foreign DNA into the MCS of phoZ produced a white
colonial phenotype on agar containing X-p and allowed discrimination between

transformants containing recombinant plasmids versus those maintaining self-annealed
or uncut vector. This cloning vector has improved the efficiency of recombinant DNA
experiments in Gram-positive bacteria.
Another method for screening and identification of recombinant clones is by using the
green fluorescent protein (GFP) obtained from jellyfish Aqueorea victoris. It is a reporter
molecule for monitoring gene expression, protein localization, protein-protein interaction.
GFP has been expressed in bacteria, yeast, slime mold, plants, drosophila, zebrafish
and in mammalian cells. Inouye et al (1997) have described a bacterial cloning vector
with mutated Aequorea GFP protein as an indicator for screening recombinant plasmids.
The pGREENscript A when expressed in E. coli produced colonies showing yellow color
in day light and strong green fluorescence under long-UV. Inserted foreign genes are
selected on the basis of loss of the fluorescence caused by inactivation of the GFP
production.
It has been observed that false results are associated while using fluorescence
technique by using GFP gene or any other reporter gene with fluorescence and color
technique using lacZ gene (Blue white screen technique) for screening and identification
of recombinant clones. Blue white screen technique gives white colonies to the
recombinant clones and blue colonies to non-recombinants. But along with it some light
blue colonies are also found. We have also observed that the white colonies may not
contain the recombinant clones. Thus this technique does not provide accurate
screening gives approximately 5-10% of false positives (Godisak R etal, Beyond pUC:
Vectors for cloning unstable DNA). This technique is cumbersome as for enhancing blue
color the plates should be kept at 40C. Also this technique requires usage of expensive
inducers like Xgal and IPTG. Similar false results have been found when GFP gene and
its variants are used as in fluorescent technique for screening and identification of
recombinant clones.
Thus the commonly used method for screening and identification of recombinant clones
are associated with problems of false positive results. Thus there is need to overcome all
the disadvantages associated with the existing techniques of screening and identification
of recombinant clones. Further there is need to provide vectors which would act both as
cloning vectors and expression vectors.

US20060099673 discloses novel recombinant gene expression method by stop codon
suppression. It describes a novel recombinant gene expression method based on a
novel recombinant gene expression vector comprising a gene encoding a selectable
marker protein which is separated by a translational stop signal from an upstream
arranged gene of interest, whereby both genes are translationally linked. Consequently,
the expression of said selectable marker gene may be reduced compared to the
expression rate of said gene of interest. It also discloses expression of said gene of
interest by using suppressor element (SECIS) in the construct to suppress the STOP
codon. Further, due to natural error rate of ribosomes the fusion protein (protein of Gene
of interest and reporter gene) is synthesized and fusion protein synthesis purely
depends on the natural error rate of the host.
However the present invention uses two STOP codons. The STOP suppression is very
much directive / dictative. The first STOP codon during selection of clones where
specific suppressor cell line is used for transformation produces fusion protein, which
aids in selection process depending on type of reporter gene used. The second STOP
codon is used mainly for authentic protein of interest in non-suppressor cell line.
Thus the present invention overcomes the disadvantages associated with the prior art by
constructing a new vector, which will make the cells fluoresce upon cloning and will be
devoid of false positive results. This way a single clone can be used for screening and
expression studies directly.
OBJECTIVE OF THE INVENTION
Accordingly one objective of the present invention is modified vector prepared by ligating
a reporter gene having a STOP codon upstream of the multiple cloning site where the
modified vector when introduced in the host cell does not fluoresce or show color
Another object of the present invention is a method for identification and screening of
recombinant clone comprising the gene of interest wherein the method involves
replacing the STOP codon in the modified vector with gene of interest having a STOP
codon different from STOP codon used with reporter gene whereby the recombinant
clones fluoresce or show color in a suitable suppression strain of the STOP codon

associated with the gene of interest and on expression in non suppressor strain do not
floroscence or show color and authentic protein of interest is obtained.
Another object of the present invention is a modified vector comprising reporter gene
having a STOP codon upstream of the multiple cloning site of the vector.
Another object of the present invention is a method of preparing a modified vector
comprising reporter gene having a STOP codon upstream of the multiple cloning site of
the vector comprising:
(a) Introduction of STOP codon upstream of multiple cloning site of the vector
(b) Amplification of reporter gene using primers
(c) Cloning of reporter gene in the vector, wherein the modified vector when
introduced in the host does not fluorescence or show color when expressed
upon induction.
Another object of the present invention is a method of preparation of recombinant clones
comprising gene of interest and modified vector wherein the method comprises:
(a) Amplification of gene of interest using specific primers containing STOP
codon other than the STOP codon used with reporter gene
(b) Cloning the amplified gene of interest in the modified vector
(c) Transformation of cloned modified vector in the STOP codon suppressor
host cell specific for STOP used in gene of interest I wherein the
recombinant clones either fluoresce or show color depending upon the
reporter gene used.
Another object of the present invention is a kit for identification and expression of
recombinant clones comprising modified vector wherein the modified vector comprise of
reporter gene carrying STOP codon.
Another object of the present invention is a kit for indicating the solubility of foreign
protein expressed using recombinant clone wherein foreign protein is expressed using a
recombinant clone identified and screened using modified vector.

SUMMARY OF THE INVENTION
Accordingly one aspect of the present invention relates to a modified vector prepared by
ligating a reporter gene having a STOP codon upstream of the multiple cloning site of
the vector whereby the modified vector when introduced in the host cell does not
floresce or show color.
According to another embodiment of the present invention there is provided a method for
identification and screening of recombinant clone comprising the gene of interest
wherein the method involves the replacing the STOP codon in the modified vector with
gene of interest having a STOP codon different from STOP codon used with reporter
gene whereby the recombinant clones fluoresce or show color in a suitable suppression
strain of the STOP codon associated with the gene of interest and on expression in non
suppressor strain do not florescence or show color and authentic protein of interest is
obtained.
According to another embodiment of the present invention there is provided a modified
vector comprising reporter gene having a STOP codon upstream of the multiple cloning
site of a vector.
According to another embodiment of the present invention there is provided a method of
preparation of a modified vector comprising a reporter gene having a STOP codon
upstream of the multiple cloning site of the vector comprising:
(a) Introduction of STOP codon upstream of multiple cloning site of the vector
(b) Amplification of reporter gene using primers
(c) Cloning of reporter gene in the vector, wherein the modified vector when
introduced in the host does not fluoresce or show color when expressed.
According to another embodiment of the present invention there is provided a method of
preparation of recombinant clone comprising gene of interest and modified vector
wherein the method comprises:

(a) Amplification of gene of interest using specific primers containing STOP
codon different from STOP codon used with reporter gene
(b) Cloning the amplified gene of interest in the modified vector
(c) Transformation of cloned modified vector in the STOP codon suppressor host
cell specific for STOP used in gene on interest wherein the recombinant
clones either fluorescence or show color depending upon the reporter gene
used.
According to another embodiment of the present invention there is provided a kit for
identification and expression of recombinant clones comprising modified vector wherein
the modified vector comprise of reporter gene carrying STOP codon.
According to another embodiment of the present invention there is provided a kit for
indicating the solubility of foreign protein expressed using recombinant clone wherein
foreign protein is expressed using a recombinant clone identified and screened using
modified vector.
Brief Description Of The Accompanying Drawings
Figure 1: Plasmid map of the pET21a vector with TAA introduction upstream of MCS
Figure 2: Restriction analysis of clones of pBAD24-GFP
Figure 3: Plasmid map of pBAD24-GFP clone (pLUBT133)
Figure 4: Release of GFP insert from pLUBT133
Figure 5: Colony PCR screening for confirmation of pET21a GFP.
Figure 6: Plasmid map of pET21a-GFP clone (pLUBT166)
Figure 7: Colony PCR screening for confirmation of modified pET21a-GFP
Figure 8: Plasmid map of pET21a modified- GFP clone ((pLUBT179)
Figure 9: GFP expression in pET21a-GFP and pET21modified-GFP clones
Figure 10: Confirmation of Ndel modification in clones 5,6,7,8,20 by double digestion
with enzymes Ndel/Hindlll
Figure 11 :Plasmid map of pLUBT 189 Figure 12: pLUBT189 digested with Xbal/Hindlll
Figure 13: Confirmation of GFP fragment insertion in pBAD24 by GFP PCR.
Figure 14: Plasmid map of pLUB191
Figure 15: SAK PCR with amber stop

Figure 16: Glowing colonies are #1, 4, 8, 9, 10 etc while the non-glowing colonies are
#5,6, 18, 42 and 48.
Figure 17: PCR for SAK gene and GFP gene
Figure 18: SDS-PAGF of SAK-GFP fusion protein in non amber suppressor strain.
Figure 19: Schematic representation for construction of pLUBT191 & 196
Figure 20: Relative fluorescence intensities of fusion proteins SAK-GFP & GCSF-GFP
DETAILED DESCRIPTION OF THE INVENTION
All the vectors described in the literature and the vectors available commercially have
either the color disappearing by insertional inactivation or the fluorescence disappearing
upon cloning. These methods can generate false positives results, as disappearance of
color or fluorescence may not be completely achieved that is there could be a possibility
of background color or fluorescence.
Thus the present invention involves construction of a modified vector for screening and
identification of recombinant clones, where in the recombinant cells fluoresce or show
color and non recombinants does not fluoresce or show color. This would avoid the false
positive results associated with prior art techniques. This vector could further be used for
expression studies.
For the present invention, the vector is selected from plasmid, phage, cosmid and the
like with no particular limitation.
Vectors suitable to be used for the present invention are numerous and a list of the
vectors can be found in the art. The vectors commercially available from Stratagene,
Promega, CLONTECH, Invitrogen GIBCO Life Sciences and other companies making
expression vectors. All the vectors with bacterial promoters may be used.
Vectors particularly suitable are plasmid vectors, which include prokaryotic, eukaryotic
and viral sequences. A list of these vectors can be found in Gene Transfer and Gene
Expression: A Laboratory Manual, Ed. Kriegler, M., Stockton Press, New York (1990)
and Molecular Cloning, A Laboratory Manual, CSH Laboratory Press, Cold Spring

Harbor, N.Y. and Current Protocols in Molecular Biology, Vol. 1, Supplement 29, section
9.66, Ed. Asubel, F. M. et al., John Wiley & Sons (2001).
The modified vector relates to a vector, which is modified to include a reporter gene with
a STOP codon.
A codon is a group of three bases - A, T, C, or G - and codes for a single amino acid, the
building blocks of proteins.
A STOP codon stops translating the code when ribosome reaches the end of the protein.
STOP codons come in three different forms - TGA, TAG, and TAA. A STOP codon
signals the cell's machinery that it has reached the end of the protein and should STOP
translating the code. More preferably for the present invention the STOP codon is TAA
only with reporter gene.
Reporter gene means a gene that is not endogenously expressed in the used cell type
that is typically used to evaluate another gene, especially its regulatory region.
Expression of reporter gene changes the phenotypic characteristic of the cell that carries
the gene. Representative reporter genes are,, beta.-galactosidase gene (.beta.-gal),
luciferase gene (luc), alkaline phosphatase gene (AP), secreted alkaline phosphatase
gene (SEAP), .beta.-glucuronidase gene (GUS), All fluorescent protein gene (Green
fluorescent protein, Yellow fluorescent protein, Red fluorescent protein, Enhanced green
fluorscent protein, Orange fluorescent protein, Cyan fluorescent protein) , substituted
pnitrophenyl phosphate and their variants
Another embodiment of the present invention relates to a method for identification and
screening of recombinant clone wherein the method involves ligating a reporter gene
having a STOP codon upstream of the multiple cloning site of a vector to prepare a
modified vector. The modified vector when introduced in the host cell do not fluroscence
or show color due to the presence of STOP codon.
The reporter gene and the vector used can be any of those disclosed above and
mentioned in the prior art.

The present invention involves a modified vector comprising reporter gene having a
STOP codon upstream of the multiple cloning site of the vector.
Another embodiment of the present invention is to provide a method of preparation of
modified vector comprising reporter gene having a STOP codon upstream of the multiple
cloning site of a vector comprising:
(a) Introduction of STOP codon upstream of multiple cloning site of the vector
(b) Amplification of reporter gene using primers
(c) Cloning of reporter gene in the vector, wherein the modified vector when
introduced in the host does not fluorescence or show color after induction.
In another embodiment of the present invention there is provided a method of
preparation of recombinant clone comprising gene of interest and modified vector
wherein the method comprises:
(a) Amplification of gene of interest using specific primers containing STOP
codon different from the one used in modified vector
(b) Cloning the amplified gene of interest in the modified vector
(c) Transformation of cloned modified vector in the suppressor host cell specific
to the STOP codon used with the gene of interest wherein the recombinant
clones either fluorescence or show color depending upon the reporter gene
used.
After identification of the recombinant clones, these recombinant clones were expressed
using a non suppressor host cell. The recombinant clones does not fluorescence and
show color and protein of interest is expressed.
The present invention involves introduction of a STOP codon upstream of multiple
cloning site of the vector using site directed mutagenesis (SDM) primers wherein one of
the codon was replaced with STOP codon. Any of the previously mentioned STOP
codon can be used. STOP codon incorporation was confirmed using DNA sequence
analysis. The most preferable STOP codon is TAA codon. The site directed mutagenesis
could be performed by any of the methods known in the art.

The next step involves cloning the reporter gene in the vector to get the modified vector.
First the reporter gene was amplified by using PCR technique and cloned into vector
carrying STOP codon. The most preferred reporter gene for the present invention is GFP
gene or beta.-galactosidase gene. The cloned modified vetcor i.e the transformants were
transformed in the host cell and the clones were examined for the presence of GFP
insert by digestion and PCR. Also this reporter gene was inserted in the non-modified
vector. The results indicate that the STOP codon interfered with GFP translation in
modified vector whereas GFP translation occurred in non-modified vector. Thus the
recombinant clones from the modified vector did not show florescence and in case of
non-modified vector showed fluorescence under UV light radiation.
For cloning any foreign gene in bacterial expression system Ndel is the preferred
restriction site as it provides start codon and avoids addition of extra amino acids at N
terminus. In the constructed modified vector, there are two Ndel sites, one is present in
MCS and required for cloning foreign gene and the other in GFP gene, which will
interfere with the cloning strategy of foreign gene. Thus the Ndel site in the GFP gene
was altered without altering the amino acid by Site Directed Mutagenesis. This vector
along with modified Ndel site of GFP was used for cloning the gene of interest. It was
confirmed by independent experiment that modification of Ndel site did not affect the
glow of GFP.
The present invention involves a method for identification and screening of recombinant
clones comprising the gene of interest wherein the method involves replacing the
multiple cloning site of the vector and the STOP codon in the modified vector with gene
of interest having a STOP codon different from the one used with reporter gene. The
above vector comprising the gene of interest when introduced in the suppressor strain
specific to the STOP codon used with the gene of interest fluoresce or shows color but
when the identified recombinant clones are introduced in the suppressor cells for
expression does not fluorescence or show color and authentic protein of interest is
obtained.
The present invention involves the use of gene of interest known to the person skilled in

the art at the time of invention.
It involves the cloning of foreign gene into the above-modified vector. Any gene of
interest can be used. The present invention offers a cost effective process for screening
and identification of recombinant clones comprising gene of interest. To exemplify the
present invention Staphylokinase gene (SAK) was cloned.
Cloning of Staphylokinase gene carrying STOP codon different from STOP codon used
with reporter gene at Ndel/EcoRI site cf the modified vector to produce a GFP fusion
protein. The most preferable STOP codon is TAG amber codon.
If the SAK gene got inserted in right frame the recombinants upon transfer to amber
suppressor strains (Since TAG amber codon is used) would glow and could be selected.
When introduced in non-Amber suppressor strains, they would make only recombinant
Staphylokinase and would not fluroscece or show color. The use of suppressor strains
would depend upon the type of STOP codon used with the gene of interest.
According to another embodiment of the present invention there is provided a kit for
identification and expression of recombinant clones comprising modified vector wherein
the modified vector comprise of reporter gene carrying STOP codon.
The modified vector according to the present invention is advantageously combined in a
kit of parts (preferably, in a cloning and expression kit) with reporter gene and carrying a
STOP codon.
A STOP codon stops translation of the code when ribosomes reach the end of the
protein. STOP codons come in three different forms - TGA, TAG, and TAA. A STOP
codon signals the cell's machinery that it has reached the end of the protein and should
stop translating the code. Any of the STOP codon can be used. More preferably for the
present invention the STOP codon used with the reporter gene to construct a modified
vector is TAA and the STOP codon used with the gene of interest is the TAG.
The reporter genes may be ,. beta.-galactosidase gene (.beta.-gal), luciferase gene
(luc), alkaline phosphatase gene (AP), secreted alkaline phosphatase gene (SEAP),

beta.-glucuronidase gene (GUS), All fluorescent protein gene (Green fluorescent
protein, Yellow fluorescent protein, Red fluorescent protein, Enhanced green fluorescent
protein, Orange fluorescent protein, Cyan fluorescent protein),substituted p-nitrophenyl
phospahte phosphate and their variants. Preferably green fluorescent protein gene
(GFP) is used.
The kit of the present invention further comprise of gene of interest carrying a STOP
codon different from STOP codon used with reporter gene. Any of the gene of interest
mentioned in the prior art can be used.
This kit can be used for:
(1) False positives could be completely avoided
(2) Kit would provide a cost effective way of screening, identification and expression of
the recombinant clones in two different bacterial cell lines
(3) This kit utilizes the property of colour or fluorescence to be obtained after cloning.
(4) This kit could be applicable to cloning of any size genes since reporter gene esp GFP
is known to fluoresce when cloned as fusion protein with any size gene at the N
terminus. #
(5) This kit would save a great deal of time since only fluorescent clones (which are
indicative of only recombinants) need to be processed for DNA preparation and
expression studies thereon.
According to another embodiment of the present invention there is provided a kit for
indicating the solubility of foreign protein expressed using recombinant clone wherein
foreign protein is expressed using a recombinant clone identified and screened using
modified vector.
It has been found that the intensity of GFP fluorescence is dependent on the solubility of
GFP. It is brightest when expressed in soluble form and decreases with decrease in
solubility. (Davis and Vierstra, 1998 ). Hence, the solubility of the protein of interest
would have an effect on the solubility of fusion protein and thereby affect the GFP
fluorescence intensity. Thus from the relative fluorescence intensity of the fusion protein
one can qualitatively assess the solubility propensity of the protein of interest.

This is applicable to all other reporter gene.
Earlier reports have suggested that GFP expression is affected by OmpT proteases as
there are two ompT protease sites in the GFP gene (Shi and Su, 2001). OmpT
expression is reported to be low at 28 deg and hence GFP expression could be more
pronounced at this temperature (Stathopoulous et al, 1999, Ogawa et al, 1995). There
are also reported inhibitors of OmpT like zinc chloride and copper chloride at 0.1 to 0.5
mM final concentration (Baneyx and Georgieu, 1990).
LE 392 is an amber suppressor strain and is known to express Ion protease and OmpT
protease. To minimize the expression of these proteases which otherwise might interfere
with GFP stability; we decided to use LE392 to express GFP with the following
conditions after transformation.
After introduction of the foreign genes like SAK, the ligation mix was introduced into
competent LE392 cells and then the plates were incubated at 30 deg C instead of
regular 37 deg C.
The preferred embodiments of the present invention are described with reference to the
following non-limiting example:
GFP gene was expressed from a known 11 expression vector. STOP codon was
introduced before the GFP gene in this vector, which resulted in non-glowing
transformants but gave positive PCR for GFP. Then Nde1 site in GFP gene was
modified by site directed mutagenesis (SDM) where as Ndel site in the vector was
available for cloning the foreign gene. This construct was used to clone foreign gene that
carried Amber STOP codon at 3' end and was cloned at Ndel site at 5' of GFP gene.
The GFP fragment along with MCS and necessary changes was subcloned in pBAD24
vector. This construct has inducible promoter which can be induced by relatively cheaper
inducer for protein expression. Amber suppressor cell line like DH5 alpha, JM109 and
LE392 were transformed with this construct. Recombinants were screened by checking
for the presence of glow under UV light and were then inoculated for DNA preparation.
These DNAs were introduced into nonamber suppressor strains like BL21 series and
then induced with the inducer to get the native protein of right size due to recognition of

amber codon as a STOP codon in the current cell line. This way a single clone can be
used for screening and expression studies directly.
Example 1: Introduction of STOP Codon into the MCS of pET21a vector:
(PLUBT176)
The STOP codon is of three types TAA, TAG and TGA. For this particular example TAA
as a STOP codon is used. But the present invention can also be carried out using any
other STOP codon and any other vector known in the prior art.
The MCS of the pET21 a vector from Novagen, USA is as follows:
RBS
AAGGAGATATA CAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT
CGC GGA TCC GAA TTC GAG CTC CGT CGA CAA GCT TGC GGC CGC ACT CGA
GCA CCA CCA CCA CCA CCA CTG A SEQUENCE ID 1
The STOP codon was introduced into pET21a vector at the base (indicated as
underlined) using the SDM primers LUBT168 and 169 by modifying the CAA to TAA.
The sequence is as follows
LUBT168: SEQUENCE ID 2
5' T AGC ATG ACT GGT GGA CAG TAA ATG GGT CGC GGA TCC GAA TTC GA 3'
LUBT169: SEQUENCE ID 3
5' TC GAA TTC GGA TCC GCG ACC CAT TTA CTG TCC ACC AGT CAT GCT A 3'
Site Directed Mutagenesis (SDM) was performed by using the Stratagene kit by using
known methods. The SDM conditions were as follows: Initial denaturation at 95oC for 30
sec followed by 15 cycles of 95oC for 30 sec, 55oC for 1 min and 68oC for 6 min as per
manufacturers instructions. After the SDM, the PCR reaction was subjected to Dpn1
digestion for 1 hr at 37 deg C. The reaction mixture was inactivated at 65 deg C for 20
minutes, cooled and then introduced into competent DH5 alpha cells. TAA incorporation
was confirmed by DNA sequence analysis of selected transformants. Figure 1 shows the
plasmid map of TAA introduction into the MCS of pET21a vector

Example 2: Cloning of Reporter gene into pBAD24 vector: (pLUBT133)
Reporter gene such as , beta-galactosidase gene (.beta.-gal), luciferase gene (luc),
alkaline phosphatase gene (AP), secreted alkaline phosphatase gene (SEAP), .beta.-
glucuronidase gene (GUS), All fluorescent protein gene (Green fluorescent protein,
Yellow fluorescent protein, Red fluorescent protein, Enhanced green fluorescent protein,
Orange fluorescent protein, Cyan fluorescent protein) can be used. Any other reporter
gene known in the prior can also be used. But for the present example, GFP gene is
used as the reporter gene.
The GFP gene was PCR amplified using primers LUBT 75/LUBT 76 the sequences of
which are given below:
LUBT 75: SEQUENCE ID 4
5' TCC CCC ATG GTA CCC GGG AGT AAA GGA GAA GAA CTT TTC ACT 3'
LUBT 76: SEQUENCE ID 5
5" CCG CCG GTC GAC CTG CAG TTA TTT GTA GAG CTC ATC CAT GCC 3'
The PCR amplification was performed with Taq DNA polymerase from Bangalore Genei
Pvt. Ltd, Bangalore, India with following amplification conditions: After an initial
denaturation time of 5 min @ 950C, 30 cycles consisting of 950C for 30 sec, 500C for 30
sec and 720C for 30 sec were continued. The amplified GFP gene was purified, digested
with Smal/Pstl and cloned into pBAD24 vector (National Institute of Genetics, Japan) at
similar sites. Figure 2 shows restriction analysis of clones of pBAD24-GFP.
The transformants were directly inoculated for plasmid DNA preparation and the DNA
from the clones were examined for the presence of GFP insert by digestion with suitable
restriction enzymes.
Example 3: Subcloning of GFP as EcoR1/Hindlll fragment in pET21a and pET21a
modified vector: (pLUBT166 and pLUBT179 respectively)
GFP was excised from pLUBT133 (Figure 3) as EcoR1/Hindlll (Figure 4 shows release
of GFP insert from pLUBT133) and cloned into pET21a (Figure 6) and modified pET21a
(Figure 8) (with STOP codon TAA in MCS) into similar sites.

The clones of pET21a-GFP (Figure 5) and pET21a modified-GFP (Figure 7)were
screened for GFP PCR. and DNAs of clones showing positive PCRs were introduced
into T7 RNA polymerase expressing cells BL21A1 (Invitrogen, USA). The cells were
then plated on LB/amp arabinose plates since arabinose acts as inducer for the T7 RNA
polymerase in BL21 A1 cells.
It was found that pET21a-GFP shows florescence but pET21a-modified-GFP does not
show florescence due to the presence of STOP codon (Figure 9). The results indicate
that the STOP codon TAA interfered with the GFP translation as expected and hence
the colonies transformed with this DNA did not show any fluorescence while the same
fragment in usual pET21a showed the glow under UV light.
Example 4: Ndel site modification of the GFP gene by SDM
Site Directed Mutagenesis was done tc modify the Ndel site which is present in the GFP
gene and interfere with the cloning strategy of foreign gene. Thus the Ndel site in GFP
gene of pLUBT179 clone was mutated by SDM using primers LUBT 188/189.
LUBT 188: SEQUENCE ID 6
5' GC TTT TCC CGT TAT CCG GAT CAC ATG AAA CGG CAT GAC3'
LUBT 189: SEQUENCE ID 7
5' GTC ATG CCG TTT CAT GTG ATC CGG ATA ACG GGA AAA GC 3"
SDM cycle
95°C - 30sec
95°C - 30 sec
55°C-1 min 15 cycles
68°C - 6 min
After SDM the ligation reaction mixture was introduced into DH5a competent cells to get
modified pLUBT179 and transformants were confirmed by linearization with Ndel. The
clones were further confirmed by double digestion with Ndel/Hindlll (Figure 10) and the
new construct was labeled as pLUBT189 (Figure 11).
Example 4: Introduction of Ndel modified GFP into pBAD24 vector
To prepare as prokaryotic expression vector under a commonly used promoter based
system, the Ndel modified GFP under T 7 promoter was placed in vector pBAD24.

The pLUBT189 was digested with Xbal/Hindlll to release the fragment (Figure 12)
having GFP gene (770 bp). It was then purified and ligated with Nhel/Hindlll digested
pBAD24 vector and the ligation mix was used to transform DH5a competent cells.
Transformants were screened by colony PCR using GFP specific primers LUBT128
and129 as forward and reverse primer respectively. Several colonies were positive for
GFP PCR (Figure 13) but never showed any fluorescence in the presence of the inducer
LUBT 128: forward primer for GFP screening- SEQUENCE ID 8
5' CCG CCG GAA TTC GTA CCC GGG AGT AAA GGA GAA GAA CTT TTC 3'
LUBT 129: reverse primer for GFP screening- SEQUENCE ID 9
5' CCG CCG GAA TTC TTA TTT GTA GAG CTC ATC CAT GCC 3'
The construct was designated as pLUBT191 (Figure 14), has GFP under arabinose
promoter but with TAA STOP at the N terminus giving a clone of GFP, which does not
glow even in the presence of inducer.
Example 5: Cloning of foreign genes into the above vector:
Any foreign gene can be used for cloning in the modified vector. For the present
example GFP-STOP vector (LUBT 191) was used. The foreign gene used in the present
example is Staphylokinase gene. However any other reporter gene containing vector
can also be used for cloning foreign gene.
Cloning of Staphylokinase gene (SAK gene) carrying Amber STOP codon at 3'end at
Ndel/EcoRI site of the modified vector was carried out. If the SAK gene got inserted in
right frame, the recombinant clones upon transfer to amber suppressor strains glow
under UV and were screened and selected. When recombinant clone was introduced in
nonamber suppressor strains, the clones do not glow and expresses only recombinant
Staphylokinase. The step for cloning foreign gene is as follows.
Staphylokinase gene was amplified from synthetic genes using specific primers
containing amber STOP codons and cloned into pLUBT191 as Ndel/EcoRI fragments.
Primers for amplification:
The primers for foreign genes must have the amber supressor codon
LUBT 009: Forward primer for Staphylokinase gene- SEQUENCE ID 10
5' CCG CCG GAA TTC CAT ATG TCA AGT TCA TTC GAC AAA GGA 3'
LUBT 187: Reverse primer for Staphylokinase gene with amber STOP 5' -
SEQUENCE ID 11

CCG CCG GAA TTC AAG CTT CTA TTT CTT TTC TAT AAC AAC 3'
PCR conditions:
The Staphylokinase gene was PCR amplified using Taq DNA polymerase from
Bangalore Genei Pvt. Ltd (Bangalore, India) with the following amplification conditions.
Initial denaturation of 4 minutes at 94 deg c followed by 30 cycles of 94oC for 30 sec,
57oc for 30 sec and 72oC for 30 sec. After a final extension of 7 min at 72 oC, the PCR
amplified product was checked on 1% agarose gel (Figure 15), purified and then
digested with Ndel/EcoRI and ligated to the GFP STOP vector pLUBT191 at similar
sites.
The ligation mix of pLUBT191 and Ndel/EcoRI digested SAK fragments were
transformed into competent LE392 cells which is an established amber suppressor strain
with the following genotype glnV44 SupF58 (lacY1 or AlacZY) galK2 galT22 metB1
trpR55 hsdR514(rK -mK +).
The transformants were replica plated on LB agar plates containing 100ug/ml ampicillin
and 13mM L(+) Arabinose.
After overnight incubation at 30oC, plate was exposed under UV and some of the
colonies were fluorescing upon UV exposure compared to others (Figure 16). The
glowing colonies are the clones of pl_UBT191-Sak.
In order to confirm that the glowing colonies are recombinant clones and non-glowing as
non-recombinants, samples of the glowing and non-glowing colonies were taken and
subjected to PCR screening with GFP specific primers as well as Staphylokinase
specific primers.
It was found that the glowing colonies give both the SAK specific and GFP PCR positive
which are due to recombinant clones and non-recombinants, which do not glow under
UV, show only GFP PCR but do not glow since they have TAA STOP codon at the N
terminus of the GFP gene. Thus 5 glowing and 5 non-glowing colonies were selected
and subjected to PCR with SAK specific and GFP specific primers.

The glowing cells are PCR positive with respect to SAK (Figure 17) (Panel A, lanes 2-6)
and non-glowing colonies are SAK PCR negative (Figure 17) (Panel A, lanes 7-11). On
the other hand all the colonies are positive for GFP PCR (Figure 17) (Panel B).
Thus it was found that when the modified constructed vector pLUBT191 under arabinose
promoter as a cloning vehicle was used, as a tool for foreign gene cloning so that only
the recombinant clones would glow. Due to insertion of foreign genes in Ndel/EcoRI site,
the STOP codon TAA gets removed and clones were selected directly based upon
fluroscence or any other without the necessity of doing PCR, restriction analysis etc.
Example 6 : Expression of the SAK-GFP fusions in non amber suppressing E. coli
strain BL21
The SAK and GFP glow positive clones # 4 and #8 along with a non-glowing #5 clone
DNA's were introduced in BL21(DE3) cells (a non amber suppressor E. coli B strain) and
expression of SAK was induced with 13mM L(+) arabinose. This result in expression of
intact SAK gene alone in these cells, since SAK is cloned with amber STOP as GFP
fusion. Expression of the heterologous proteins was analyzed on sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the protein synthesis
terminated at amber STOP and proteins of molecular size (15 kDa) was observed on
SDSPAGE (Figure 18).
The non-glowing clone did not express SAK since it is a non-recombinant with TAA at
the N terminus of the GFP gene in pLUBT191
The applications of the present invention includes having following advantages as:
(1) The modified vector of the present invention provides consistent and reliable
results for identification and screening of recombinant clones as they show
colour or fluorescence after cloning whereas all other commercially available
vectors show loss of colour or loss of fluorescence on cloning.
(2) In the present invention recombinants fluoresce under UV light or show color
wherein non-recombinant does not fluoresce or color when introduced in the
suppressor strain.
(3) False positives are completely avoided which is not possible with the
commercially available vectors.

(4) Any other reporter gene found in the prior art could be used in place of GFP
gene in the same vectors or different vectors.
(5) Different inducers can be used based upon the types of promoters used.
(6) The present invention also uses same vector for screening and expression in
two different bacterial cell lines.
(7) The present invention can be used for cloning of any size genes since GFP is
known to fluoresce when cloned as fusion protein with any size gene at the N
terminus.
(8) The ligation mix using this vector need not be dephosphorylated as done in
prior art since self ligated vector molecules without insert would never glow
under UV light.
(9) This is a cost-effective process since it is not necessary to run control ligation
(that is ligation with vector alone) at all, since only recombinants would glow
and all our experiments show that this control is absolutely tight and not
leaky.

(10) This vector would be cost-effective since it avoids the cost of restriction
analysis, PCR etc which are routinely used for screening recombinants.
(11) This is a time saving method since only fluorescense or observation of
color will differentiate between recombinant clones and non-recombinants.
(12) Further, Since GFP fluorescence is brightest when it is expressed in
soluble form, the intensity of the fluorescence after cloning the foreign gene
also indicates the extent of solubility of the fusion protein. This is the report of
the first vector, which will indicate solubility of the foreign gene based on
intensity of fluorescence.
Example 6: Qualitative assessment of the relative solubility of the proteins of
interest
The solubility of the protein of interest would have an effect on the solubility of fusion
protein and thereby affect the GFP fluorescence intensity. Thus from the relative
fluorescence intensity of the fusion protein one can qualitatively assess the solubility
propensity of the protein of interest. To validate this hypothesis, hGCSF was cloned
(produced as insoluble aggregates in E coli) and SAK (produced as soluble protein in E.
coli) as GFP fusions with amber STOP. Both the constructs were introduced into
competent LE392 E. coli cells (an amber suppressor strain) and plated on LB-agar semi-

solid media containing 100g/ml ampicillin and 13mM L(+) arabinose. Upon UV
exposure a difference in fluorescence intensity depending on the solubility of the fusion
protein was found. The results showed that (Figure 20), SAK-fusion glow is brighter than
hGCSF- fusion. This is due to better solubility of SAK protein over hGCSF.
SEQUENCE LISTING
<110> Lupin LIMITED
<120> Vector for identification, selection and expression of
recombinants
<130> PCC 3985
<140>
<141>
<150>
<151>
<160> 11
<170> Patentln version 3.5
<210> 1
<211> 117
<212> DNA
<213> Artificial Sequence
<220>
<221>: misc_feature
<222>:
<223>: Multiple Cloning Sites of the pET21a vector
<400> 1
aaggagatat acatatggct agcatgactg gtggacagca aatgggtcgc ggatccgaat 60
tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactga 117
<210> 2
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<221>: Primer

<222>:
<223>: SDM primers LUBT168
<400> 2
tagcatgact ggtggacagt aaatgggtcg cggatccgaa ttcga 45
<210> 3
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<221>: Primer
<222>:
<223>: SDM primers LUBT169
<400> 3
tcgaattcgg atccgcgacc catttactgt ccaccagtca tgcta 45
<210> 4
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<221>: Primer
<222>:
<223>: LUBT 75 primer of GFP gene
<400> 4
tcccccatgg tacccgggag taaaggagaa gaacttttca ct 42
<210> 5
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<221>: Primer
<222>:
<223>: LUBT 76 primer for GFP gene
<400> 5
ccgccggtcg acctgcagtt atttgtagag ctcatccatg cc 42
<210> 6
<211> 38

<212> DNA
<213> Artificial Sequence
<220>
<221>: Primer
<222>:
<223>: LUBT 188 primer
<400> 6
gcttttcccg ttatccggat cacatgaaac ggcatgac 38
<210> 7
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<221>: Primer
<222>:
<223>: LUBT 189 primer
<400> 7
gtcatgccgt ttcatgtgat ccggataacg ggaaaagc 38
<210> 8
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<221>: Forward Primer
<222>:
<223>: LUBT 128 forward primer for GFP screening
<400> 8
ccgccggaat tcgtacccgg gagtaaagga gaagaacttt tc 42
<210> 9
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<221>: Reverse Primer
<222>:
<223>: LUBT 129 reverse primer for GFP screening
<400> 9

<210> 10
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<221>: Forward Primer
<222>:
<223>: LUBT 009 Forward primer for Staphylokinase gene
<400> 10
ccgccggaat tccatatgtc aagttcattc gacaaagga 39
<210> 11
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<221>: Reverse Primer
<222>: amber STOP 5'of Staphylokinase gene
<223>: LUBT 187 Reverse primer for Staphylokinase gene with amber STOP 5'
<400> 11
CCGCCGGAAT TCAAGCTTCT ATTTCTTTTC TATAACAAC

WE CLAIM:
1. A modified vector prepared by ligating a reporter gene having a STOP codon
upstream of the multiple cloning site of the vector.
2. A modified vector according to claim 1 wherein the modified vector do not
floresce or show color in presence of inducer.
3. A modified vector as in claim 1 wherein STOP codon used may be selected from
TAA, TAG or TGA.
4. A modified vector as in claim 1 wherein reporter genes used may be ,. beta.-
galactosidase gene (.beta.-gal), luciferase gene (luc), alkaline phosphatase gene
(AP), secreted alkaline phosphatase gene (SEAP), .beta.-glucuronidase gene
(GUS), All fluorescent protein gene (Green fluorescent protein, Yellow
fluorescent protein, Red fluorescent protein, Enhanced green fluorescent protein,
Orange fluorescent protein, Cyan fluorescent protein) , substituted p-nitrophenyl
phosphate and their variants.
5. A method for identification and screening of recombinant clone comprising the
gene of interest wherein the method involves the replacing the multiple cloning
site and the STOP codon in the modified vector with gene of interest having a
STOP codon different from STOP codon used with reporter gene whereby the
recombinant clones florescence or show color in a suitable suppressor strain of
the STOP codon associated with the gene of interest.
6. A method for identification and expression of recombinant clone as in claim 5
wherein STOP codon may be selected from TAA, TAG or TGA.
7. A method for identification and expression of recombinant clone as in claim 5
wherein reporter genes may be ,. beta.-galactosidase gene (.beta.-gal),
luciferase gene (luc), alkaline phosphatase gene (AP), secreted alkaline
phosphatase gene (SEAP), .beta.-glucuronidase gene (GUS), All fluorescent
protein gene (Green fluorescent protein, Yellow fluorescent protein, Red
fluorescent protein, Enhanced green fluorescent protein, Orange fluorescent
protein, Cyan fluorescent protein), substituted p-nitrophenyl phosphate and their
variants.
8. A method for identification and expression of recombinant clone as in claim 5
wherein the recombinant are free from false results.

9. A method for identification and expression of recombinant clone as in claim 5
where in the selected recombinant clones when expressed in the non-suppressor
strain do not fluoresce or show color and give recombinant protein expression.
10. A modified vector comprising reporter gene having a STOP codon upstream of
the multiple cloning site of the vector.
11. A modified vector as in claim 10 wherein STOP codon may be selected from
TAA, TAG or TGA.
12. A modified vector as in claim 10 wherein reporter genes may be heme binding
part of cytocrome (part of kit, Eurogenetec),. beta.-galactosidase gene (.beta.-
gal), luciferase gene (luc), alkaline phosphatase gene (AP), secreted alkaline
phosphatase gene (SEAP), .beta.-glucuronidase gene (GUS), All fluorescent
protein gene (Green fluorescent protein, Yellow fluorescent protein, Red
fluorescent protein, Enhanced green fluorescent protein, Orange fluorescent
protein, Cyan fluorescent protein)), substituted p-nitrophenyl phosphate and their
variants.
13. A modified vector as in claimiO wherein vector is selected from plasmid, cosmid
and phage.
14. A method of preparation of a modified vector comprising a reporter gene having
a STOP codon upstream of the multiple cloning site of a vector comprising:

(a) Introduction of STOP codon upstream of multiple cloning site of the vector
(b) Amplification of reporter gene using primers
(c) Cloning of reporter gene in the vector
Wherein the modified vector when introduced in the host does not fluoresce or
show color when induced.
15. A method of preparation of a modified vector as in claim 14 wherein STOP codon
may be selected from TAA, TAG or TGA.
16. A method of preparation a modified vector as in claim 14 wherein reporter genes
may be ,. beta.-galactosidase gene (.beta.-gal), luciferase gene (luc), alkaline
phosphatase gene (AP), secreted alkaline phosphatase gene (SEAP), .beta.-
glucuronidase gene (GUS), All fluorescent protein gene (Green fluorescent
protein, Yellow fluorescent protein, Red fluorescent protein, Enhanced green
fluorescent protein, Orange fluorescent protein, Cyan fluorescent protein)
substituted p-nitrophenyl phosphate and their variants.

17. A method of preparation of recombinant clone comprising gene of interest and
modified vector wherein the method comprises:
(a) Amplification of gene of interest using specific primers containing STOP
codon different from STOP codon used with reporter gene
(b) Cloning the amplified gene of interest in the modified vector
(c) Transformation of cloned modified vector in the STOP codon suppressor
host cell wherein the STOP codon suppressor host cell is specific for STOP
codon used with the gene of interest where in the recombinant clones
either fluorescence or show color depending upon the reporter gene used.

18. A method of preparation a recombinant clone as in claim 17 wherein STOP
codon may be selected from TAA, TAG or TGA different from STOP codon used
with reporter gene.
19. A method of preparation a recombinant clone as in claim 17 wherein reporter
genes may be,, beta.-galactosidase gene (.beta.-gal), luciferase gene (luc),
alkaline phosphatase gene (AP), secreted alkaline phosphatase gene (SEAP),
.beta.-glucuronidase gene (GUS), All fluorescent protein gene (Green fluorescent
protein, Yellow fluorescent protein, Red fluorescent protein, Enhanced green
fluorescent protein, Orange fluorescent protein, Cyan fluorescent protein) ,
substituted p-nitrophenyl phosphate and their variants.
20. A method of preparation of recombinant clone as in claim 19 wherein the
recombinant clones when expressed in non suppressor host cell do not fluoresce
or show color & yields protein of interest.
21. A kit for identification and expression of recombinant clones comprising modified
vector wherein the modified vector comprises of reporter gene carrying STOP
codon.
22. A kit for identification and expression of recombinant clones wherein as in claim
21 STOP codon may be selected from TAA, TAG or TGA.
23. A kit for identification and expression of recombinant clones wherein as in claim
21 reporter genes may be ,. beta.-galactosidase gene (.beta.-gal), luciferase
gene (luc), alkaline phosphatase gene (AP), secreted alkaline phosphatase gene
(SEAP), .beta.-glucuronidase gene (GUS), All fluorescent protein gene (Green
fluorescent protein, Yellow fluorescent protein, Red fluorescent protein,
Enhanced green fluorescent protein, Orange fluorescent protein, Cyan
fluorescent protein), substituted p-nitrophenyl phosphate and their variants.

24. A kit for identification and expression of recombinant clones wherein as in claim
21 further comprise of gene of interest carrying a STOP codon different from
STOP codon used with reporter gene.
25. A kit for indicating the solubility of foreign protein expressed using recombinant
clone wherein foreign protein is expressed using a recombinant clone identified
and screened using modified vector.

A modified vector prepared by ligating a reporter gene having a STOP codon upstream
of the multiple cloning site of the vector. A method for identification and screening of
recombinant clone comprising the gene of interest wherein the method involves the
replacing the multiple cloning site and the STOP codon in the modified vector with gene
of interest having a STOP codon different from STOP codon used with reporter gene
whereby the recombinant clones florescence or show color in a suitable suppressor
strain of the STOP codon associated with the gene of interest. A modified vector
comprising reporter gene having a STOP codon upstream of the multiple cloning site of
the vector A method of preparation of a modified vector comprising a reporter gene
having a STOP codon upstream of the multiple cloning site of a vector comprising
Introduction of STOP codon upstream of multiple cloning site of the vector; Amplification
of reporter gene using primers; cloning of reporter gene in the vector, wherein the
modified vector when introduced in the host does not fluoresce or show color when
induced,. A method of preparation of recombinant clone comprising gene of interest and
modified vector wherein the method comprises amplification of gene of interest using
specific primers containing STOP codon different from STOP codon used with reporter
gene; Cloning the amplified gene of interest in the modified vector; transformation of
cloned modified vector in the STOP codon suppressor host cell wherein the STOP
codon suppressor host cell is specific for STOP codon used with the gene of interest
where in the recombinant clones either fluorescence or show color depending upon the
reporter gene used. A kit for identification and expression of recombinant clones
comprising modified vector wherein the modified vector comprises of reporter gene
carrying STOP codon.