F0RM2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. Title of the Invention: LETHAL GENE AS A TOOL FOR ZERO
BACKGROUND CLONING

2. Applicant(s) (a) NAME :
(b) NATIONALITY:
(c) ADDRESS :

LUPIN LIMITED
An Indian Company.
159, CST Road, Kaiina, Santacruz (East), Mumbai-400 098, Maharashtra, India.

3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed:


Field of invention:
The present invention relates to a lethal gene DNA fragment which comprises the nucleotide sequence represented by SEQ ID NO: 1. The invention also relates to a dual purpose cloning and expression vector comprising LG gene of SEQ ID NO. l.The invention further relates to method of producing the said vector and method of cloning using the same and a kit.
Background of the invention:
Molecular cloning is an important tool 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 themselves. The steps involved in the molecular cloning of genes are as follows: (a) Choice of DNA: genomic DNA or cDNA. (b)Insertion of the chosen DNA into a vector - The cloning of DNA requires the production of large quantities of the DNA of interest. This is accomplished by taking advantage of the fact that bacteria and bacteriophages replicate their DNA with high fidelity, many times, in a relatively short time frame. There are two types of cloning vectors used routinely. The most popular vectors currently in use consist of either small circular DNA molecules (plasmids) or bacterial viruses (phage). The fragment of DNA to be amplified is first inserted into a cloning vector.
For this plasmid vector is usually prepared for ligation by first linearizing the plasmid with a restriction endonuclease. Typically, the ends of the DNA segment and the ends of the linearized vector are compatible to allow ligation using DNA ligase. Alternatively, it may be possible for a DNA segment to be ligated to circular plasmid vector using a recombinase enzyme. The ligation reaction is subsequently transformed into a suitable host cell, usually E. coli. The procedure of DNA cloning creates a new plasmid/phage, called the recombinant plasmid/phage, consisting of the vector and DNA segment, which is also denoted as the DNA insert. The recombinant plasmid/phage can be used to characterize the insert, such as by DNA sequencing, RNA transcription, transfection, or site directed mutagenesis. Alternatively, the recombinant plasmid may be useful for applications such as gene therapy.
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The fundamental problem with DNA cloning methods is the large background transformation. Background transformations are transformed cells, which contain the recircularized cloning vector without any insert, also denoted as non-recombinants. The presence of background transformants requires tedious screening for colonies that contain the recombinant plasmid.
For applications such as making DNA libraries, the presence of background transformants is detrimental to the quality of the library. It is critical to keep the background transformation low to make a quality library.
Most prior art methods of DNA cloning use vectors that dp not reduce the background transformation rate. Instead, these vectors are designed to help identify recombinant colonies among a large number of background transformants by plating on special media. For example, the commonly used pUC18 and M13mpl8 vectors use insertional inactivation of the beta-galactosidase gene to identify recombinants. In the presence of the histochemical substrate-X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactose), background clones are blue in color while recombinant clones are colorless.
The vector pBR322 uses insertional inactivation of the tetracycline gene to identify recombinants. Recombinant clones are tetracycline sensitive while background clones are tetracycline resistant. The disadvantages of this approach are (a) it requires the use of special cloning vectors, and (b) there is no reduction in the background transformation rate.
JP-A-57-139095 discloses a method, which uses a lethal gene such as a topoisomerase or coiicin El gene as the gene marker. In this method, an exogenous gene is inserted into the translation region of a lethal gene, so that expression of the gene is inhibited, and only a clone harboring the exogenous gene is selectively grown. However, in the case of selection by coloring using a .beta.-galactosidase gene or the like, not only it is necessary to add a coloring substance such as X-gal to the medium, but also transformants not harboring the insertion fragment are also grown, so that a large area of the agar medium is required for isolating a large number of transformants. On the other hand, in the case of using a lethal gene, the transformants not harboring the insertion fragment die out, so that it is possible to reduce the medium area for isolating transformants or to carry out the selection by using a liquid medium. However, when lethality of the lethal gene is too high, (1) mutation is introduced into the lethal
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gene at a high frequency during the culturing, so that the lethality cannot be maintained stably, and (2) it is necessary to use a host into which an inactivated gene or mutation is introduced, for regulating toxicity of the lethal gene in amplifying the vector. Also, when lethality of the lethal gene is low, a promoter having high expression activity is necessary for exerting the lethality by over-expression.
A zero background cloning kit (Invitrogen,) utilizes the property of a known E. coli lethal gene called ccdB in the expression vector under plac promoter. However, this plasmid has a fusion of lacZ and ccdB with MCS (Multiple cloning sites) in the middle of the lacZ gene. Any foreign insertion into this vector leads to inactivation of the fusion protein and hence only recombinants carrying the gene of interest grows. This vector has certain disadvantages. This vector could be useful only for cloning purposes and cannot be used for expression studies since after expression the foreign protein would be a lacZ fusion and hence the foreign gene would not have the native N terminal sequence.
Another method relates to development of a vector with GFP as the gene in the vector, which upon insertion of the foreign gene would not fluoresce under UV light.
Further WO 200015846 discloses labeled DNA segment and introducing it into the vector and transforming the labeled DNA using Biotin into host cell. The invention is to provide a method for reducing the background transformation in DNA cloning which works with any vector and any DNA segment to be cloned. However this method suffers from certain disadvantages, as the method is tedious, requires a high amount of vector DNA, used only for cloning the gene of interest and requires an experienced person to perform.
Thus there is need to overcome ail the disadvantages associated with prior art vectors and method of cloning and providing a zero background cloning. Further there is need to provide vectors which would act both as expression as well as cloning vectors
The present invention aims at preparing dual purpose LG containing vector used for cloning and expression studies. A BLAST search indicated no similarity of the LG amino acid sequence with any sequence available in the database.
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This novel vector gives essentially >95% recombinants as successful clones carrying the gene of interest. The vector can be used successfully to clone DNA fragments of any size and does not require any special bacterial host for expression studies.
This vector could be used for lysis of the bacterial cells and hence could be used to avoid bacterial lysis in large scale, which would prove to be a cost-effective process. No antidote for this lethal gene would assure killing of strains completely.
Objectives of the present invention:
Accordingly an object of the present invention is to provide a Lethal gene DNA fragment (LG gene) which comprises the nucleotide sequence represented by SEQ ID NO: 1.
Another object of the present invention is the use of lethal gene DNA fragment of SEQ ID NO: 1 for cloning and expression purpose.
Another objective of the present invention is to provide a dual purpose vector (s) comprising the lethal gene of SEQ ID NO: 1 for cloning and expression
Another objective of the present invention is to provide an expression and cloning vector comprising a Lethal Gene DNA sequence wherein said Lethal Gene containing vector comprising: a) a Lethal Gene encoding 53 amino acid comprising of SEQ ID NO: 1 capable of being iigated with vector, b) a cloning site upstream of the lethal gene for the gene of interest to be introduced into Lethal Gene containing vector.
Another object is to provide a process for preparation of the dual-purpose cloning and expression vector.
Another objective of the present invention a method of preparing a cloning and expressing DNA fragment.
Another objective of the present invention a dual purpose cloning and expression kit comprising a Lethal Gene having SEQ ID NO 1 and a vector.
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Another objective of the present invention relates to kit comprising a Lethal Gene having a SEQ ID NO 1 and a vector, which is, used for cloning and expression studies.
Summary of the invention
Accordingly, one aspect of the present invention relates to a Lethal gene comprising SEQ ID NO: 1. ATG TTT TAT ATT CCC CAG AAC ATC AGG TTA ATG GCG TTT TTG ATG TCA TTT TCG CGG TGG CTG AGA TCA GCC ACT TCT TCC CCG ATA ACG GAG ACC GGC ACA CTG GCC ATA TCG GTG GTC ATC ATG CGC CAG CTT TCA TCC CCG ATA TGC ACC ACC GGG TAA
According to another embodiment of the present invention there is provided a DNA cloning and expression vector comprising;
a. lethal gene comprising SEQ ID NO: 1: ATG TTT TAT ATT CCC CAG AAC
ATC AGG TTA ATG GCG TTT TTG ATG TCA TTT TCG CGG TGG
CTG AGA TCA GCC ACT TCT TCC CCG ATA ACG GAG ACC GGC ACA
CTG GCC ATA TCG GTG GTC ATC ATG CGC CAG CTT TCA TCC CCG
ATA TGC ACC ACC GGG TAA;
b. at least one cloning site upstream of the lethal gene.
According to embodiment, the present invention relates to a method for producing a cloning and expression vector comprising lethal gene, said process comprising:
a. amplification of the lethal gene comprising SEQ ID NO:l ATG TTT TAT ATT
CCC CAG AAC ATC AGG TTA ATG GCG TTT TTG ATG TCA TTT TCG CGG TGG CTG AGA TCA GCC ACT TCT TCC CCG ATA ACG GAG ACC GGC ACA CTG GCC ATA TCG GTG GTC ATC ATG CGC CAG CTT TCA TCC CCG ATA TGC ACC ACC GGG TAA using suitable primers, and
b. cloning of the same in a cloning and expression vector at the end of MCS sequence.
Accordingly, another embodiment of the present invention relates to a kit comprising a lethal
gene of SEQ ID NO: I ATG TTT TAT ATT CCC CAG AAC ATC AGG TTA ATG GCG
TTT TTG ATG TCA TTT TCG CGG TGG CTG AGA TCA GCC ACT TCT TCC
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CCG ATA ACG GAG ACC GGC ACA CTG GCC ATA TCG GTG GTC ATC ATG CGC CAG CTT TCA TCC CCG ATA TGC ACC ACC GGG TAA and vector,.
Accordingly, another embodiment of the present invention relates to a method of cloning and expressing DNA fragment of interest comprising (a) ligating the DNA fragment of interest with vector comprising Lethal Gene of Seq ID NO: 1 ATG TTT TAT ATT CCC CAG AAC
ATC AGG TTA ATG GCG TTT TTG ATG TCA TTT TCG CGG TGG CTG AGA
TCA GCC ACT TCT TCC CCG ATA ACG GAG ACC"GGC ACA CTG GCC ATA TCG GTG GTC ATC ATG CGC CAG CTT TCA TCC CCG ATA TGC ACC ACC GGG TAA; (b) transforming into host cell; (c) cloning and expression in a medium containing inducer, wherein the Lethal Gene vector does not require dephosphorylation
Brief Description Of The Accompanying Drawings
Figure 1 shows Growth Curve BL21 (DE3) cell containing the pET21a-LG vector showing the
lethal effect of LG gene at 600nm absorbance.
Figure 2. Schematic representation of construction of pLUBT036
Figure3 shows the plasmid map of pLUBTl 12 vector.
Figure 4: Growth Curve containing the pLUBTl 12 vector showing the lethal effect of LG gene Figure 5 shows lethal effects of lethal gene in BL21 derived cell lines
Figure 6 shows (panel B in both the cases) colonies surviving on arabinose plates indicative of
successful cloning of pLUBTl 12 with GCSF and Sak genes respectively
Figure 7 (a) and 7(b): Electrophoresis bands of PCR screening of GCSF clones and release of
GCSF inserts using from LB amp and LB amp arabinose plates
Figure 8A and 8B shows the electrophoresis bands of PCR screening of SAK clones and release of SAK inserts using LB amp and LB amp arabinose plates
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Figure 9 shows photographs showing plates of colony of transformed cells, cloned using
pLUBTl 12 vector with or without dephosphorylation.. There is more than 99.99% reduction in
colony number since LG is expressed in the panel B while it is not expressed in panel A
Figure 10 shows photograph showing the killing of DH5 a cells at ImM, 10 mM, and 100 mM
cone, of ArabinoseCells with 50 ng and 400 ng of pLUBT112 DNA plated on LB agar plates
with amp and 13 mM Arabinose
Figure llphotograph showing death of DH5a cells with 50 ng and 400 ng of pLUBT112
plated on 13 mM Arabinose
Figure 12 shows Electrophoresis Bands of GST-LG Fusion Protein
Detailed description of the invention:
The present inventors have developed novel Lethal Gene (LG) having SEQ ID NO: 1 for zero background-cloning vehicles which overcomes the disadvantages associated with prior art in terms of background cloning. Surprisingly the present inventors have found that the lethal gene cloned into the last restriction site in such a way that when any exogenous gene is inserted into the Multiple cloning sites (MCS) region upstream of the lethal gene, the expression of the lethal gene is inhibited, and only clones harboring the exogenous gene survives while non-recombinants which express the lethal gene product dies. This provides zero background cloning.
The present invention relates to a Lethal gene (LG) DNA fragment which comprises of the nucleotide sequence represented by SEQ ID NO:I. Further this LG DNA fragment when introduced into a vector to prepare a dual purpose LG containing vector for cloning and expressing studies comprising a Lethal gene of SEQ ID NO:l wherein the Lethal gene of SEQ ID NO:l is ligated with vector and transforming the vector into host cell. Thus the present ,invention relates to a novel lethal gene which when ligated with vector produces zero background clones, because relegated vector carrying the intact toxic gene are lethal to host cell. For efficient positive selection the lethality of the marker gene must be strong enough completely to kill the clones harboring the vector without the insert, while for propagation, the
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same vector without the insert must be maintained stably overriding the activity of lethal gene. A better expression of lethal gene is achieved using E.coli strains coding rare codons.
The lethal gene encodes a protein having 53 amino acids and the gene product kills the host on its expression. Some E. coli colonies having recombinant pET21a vector with a ccdB gene insert in the reverse orientation died abnormally during a cloning experiment. Further investigation showed that when this clone (SEQ ID NO: 2) expressed in broth after addition of arabinose inducer, one open reading frame was coding a protein having 53 amino acids. This protein is found out to be lethal to E. coli. The lethality of the LG gene is different from that of common ccdB lethal gene. While ccdB protein has ccdA gene product as an antidote, LG have no such antidotes.
The concerned new gene was named as LG and BLAST was carried on the said gene and no match found. Further this LG gene is used for constructing a new vector named pLUBT112 containing this novel Lethal gene. This vector can be used both as a zero-background cloning vehicle and as an expression vector
The present inventors have prepared a dual-purpose vector comprising the lethal gene for use for cloning and expression studies. A BLAST search indicated no similarity of the LG amino acid sequence with any sequence available in the database.
This novel vector gives essentially >95% recombinants as successfiil clones carrying the gene of interest. The vector can be used successfully to clone DNA fragments of any size and does not require any special bacterial host for expression studies.
This vector is used for lysis of the bacterial cells and hence could prevent bacterial lysis in large scale, which would prove to be cost-effective. No antidote for this lethal gene would assure killing of strains completely.
For the present invention, the vector is selected from plasmid, phage, cosmid and the like with no particular limitation.
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The vector is successfully cloned with DNA fragments of any size and does not require any special bacterial host for expression. Preferably the size of DNA fragment of interest may vary from 3kb to lOkb.
E. coli is used as the host cell for cloning and expression of vector. Other host cells could also be used. When the transformant having a DNA of interest is selected, the selection is generally carried out by allowing the transformants to grow in a mediutn preferably LB agar having a antibiotic marker preferably ampicillin and an inducer. Preferably for the present invention the medium is LB agar plate with ampicillin and the inducer as arabinose. The present invention involves any medium or inducer known to the person skilled in the art at the time of invention. Preferably the inducer is arabinose. Inducers could be IPTG (17, tac promoters), lactose (lac promoter), tryptophan (Trp promoter), heat (42°C),15 degC(CoId shock promoter- CspA) , sodium chloride for salt inducible promoters and the like. The type of inducer used depends upon the type promoter. The present invention also includes analogues of arabinose known in the art like are D-Fucose. Thus the present invention related to construction of vector containing the LG gene and ITgating the DNA fragment of interest with LG containing vector.
This is then introduced in the host cell and cloned in agar medium containing ampicillin as antibiotic marker and arabinose as an inducer. The arabinose inducible promoter is pBAD promoter. The present invention involves any promoters known to the person skilled in the art at the time of invention. Preferably the promoter is pBAD promoter such such an effect could be achieved with alternate promoters like but not limited to T5; T7, Trp,CspA, pLac, lambda pL promoters, salt inducer promoters, wherein suitable inducers could be incorporated in the medium along with the antibiotic of choice.
When the vector of the present invention is used, transformants having no insertion fragment do not amplify and the one containing the insertion fragment grow in the medium. This novel vector gives essentially >95% recombinants as successful clones carrying the gene of interest. Thus an effective means is provided for efficiently selecting a clone having an exogenous insertion gene fraction, in carrying out transformation using a lethal gene of SEQ ID NO: 1.
Hence the present invention provides a useful means for cloning and expression of exogenous insertion gene.
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This could further be used for lysis of the bacterial cells and hence could be used to avoid bacterial lysis in large scale, which would prove to be a cost-effective process. The lethal gene under mammalian inducer promoter may also find use in killing of mammalian cells especially cancer cells.
The preferred embodiments of the present invention are described with reference to the following non-limiting examples;
Example 1
The lethal gene (LG as called here) having 53 amino acids showed lethality to E. coli when
expressed in broth after addition of the inducer.
This lethal gene was amplified using suitable primers to have only the 53 amino acid gene
length under T7 promoter based expression vector- pET21a.
The primers for Lethal gene amplification by PCR were as follows:
Forward LG primer: 5' CCG CCG GAA TTC CAT ATG TTT TAT ATT CCC CAG AAC
ATC AGG TTA 3' SEQ ID NO: 6
Reverse LG primer: 5' CCG CCG GAA TTC AAG CTT TTA CCC GGT GGT GCA TAT
CGG GGA TGA 3' SEQ ID NO: 7
PCR conditions were:
l.Cydel: 94°C for 4 minxl cycle
2.Cycle 2: 94°C for 30 sec, 58°C for 30 sec, 72°C for 30 sec x 30 cycles
3.Cycle 3: 72°C for 5 min xl cycle
1A. Cloning of pET21a-LG and expression studies
The PCR amplified product of LG (SEQ ID NO: I & Amino acid Sequence No la) was purified and digested with Ndel/HindlH and cloned into pET2Ia. The recombinants were checked for release for the LG insert by Ndel/HindlH digestion. One of the positive clones of pET21 a-LG was sequenced and the data is given in SEQ ID NO: 3. The clones of pET21a-LG were later introduced into BL21 (DE3) cells and then induced with 1 mM IPTG. Upon induction with IPTG for 2 hours at 37 deg C, the BL21(DE3) cells carrying the pET21a-LG clone was found to have dramatic reduction in cell density by measuring
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absorbance at 600nni (Figure 1) and also found to lyse which is a typical sign of bacterial death (Table 1).
Table 1:IPTG induced and Uninduced data for pET21a-LG (At absorbance 600nm)

Hrs Uninduced Induced
0 0.81 0.8
1 1.33 0.87
2 2.16 0.92
3 2.3 0.84
4 \2A 0.7
5 2.5 0.72
6 £.88 0.74
7 3.4 0.67
Figure 1 shows a growth curve obtained by plotting the absorbance data measured at 600 nm of sample containing the transformed BL21 (DE3) cells. It shows dramatically decrease in cell density on induction of LG expression. Table 1 showing increase in the cell lysis and decrease in cell density on induction with time.
Since pET21a vector requires specific cell-lines expressing T7 RNA polymerase, it was decided to have ttiis lethal gene in a bacterial vector which can be easily induced in routinely used bacterial hosts like DH5 alpha and JM109. For this purpose the most comtmonly used vector pBAD24 vector was used. Example IB
Construction of pBAD24m vector:
T7 forward and T7 reverse primer were taken and used as template for amplification from
pET21a vector. The PCR conditions were as follows:
Forward primer: 5' TAA TAC GAC TCA CTA TAG GG 3' SEQ ID NO: 8
Reverse primer: 5' GCT AGT TAT TGC TCA GCG G 3' SEQ ID NO: 9
PCR conditions: 1.95°C 5 min
2. 95°C 30 sec
3.55°C 30 sec
4. 72°C 1 min 5.Go to Step 2, 25 times
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6. 72UC 7 min
The PCR product was digested sequentially with Xbal/Hindlll and cloned into Nhel/Hindlll sites of pBAD24 vector. Thus T7 glO and the SD sequence of pET21a (65 bp) in pBAD24 vector were obtained and construction of pLUBT036 is shown in Figure 2. This vector was designated as pBAD24m (m refers to modified). Right clones would get digested with Ndel while Ndel does not cut native pBAD24 vector. The pBAD24m (pLUBT036) was sequenced with arabinose promoter primer and the sequencing data is shown in SEQ ID NO. 4 The next step is to have the LG gene in the modified pBAD24m vector with MCS at the 5' end so that suitable restriction enzyme sites could be used for cloning any gene of interest. For this purpose fresh primers were designed for PCR amplification of the LG gene under the following PCR conditions.
Example 2
Cloning of LG as Hindlll/Hindlll fragment in pLUBT036:
Forward Primer: 5' CCG CAA GCT TGC TTT TAT ATT CCC CAG AAC ATC AGG TTA
3'SEQ ID NO: 10
Reverse Primer: 5' CCG CCG GAA TTC AAG CTT TTA CCC GGT GGT GCA TAT CGG
GGA TGA 3' SEQ ID NO:l 1
PCR conditions were identical to the one done for pET21a cloning.
LG clone was constructed in pLUBT036 (SEQ ID NO 4) at the Hind III site (restriction site)
Clones were screened by replica plating on LB ampicillin plate with arabinose (13 mM final
concentration) and subsequently restriction digested with H3. Lethal effects were studied in
various E.coli strains on LB-Amp and LB-Amp-Arabinose plates with pLUBT112 (construct
with LG at Hindlll site). Results indicate that MCS does not affect lethal properties of LG and
hence this could be a potential cloning vehicle. Plasmid map of pLUBT112 is explained in
Figure 3 and the data of sequencing is shown in SEQ ID NO:. 5
Example 3
Expression of LG from pLUBT112 in DH5 alpha cells in liquid medium:
DH5 alpha cells were transformed with pLUBT112 and induced in LB agar with 13 mM Arabinose and absorbance was read at 600 nm at different hours post induction. Results indicate that the growth of induced cells is much lower than uninduced cells (Figure 4, Table
2)
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Table 2: Data for growth of arabinose induced and Uninduced cells

Absorbance at 600nm Uninduced (A600) [nduced (A600)
At induction 0.44 0.44
1 hr 2.62 1.18
2hr 3.88 0.88
3hr 3.50 0.90
Thus LG expression stops the growth of E.coli DH5 alpha cells and hence this gene is bacteriostatic to E. coli.
LG action appears to be different than ccdB action
It is known that ccdB action is known to be regulated by ccdA in the E. coli host. It was found that LG indeed is lethal even to cells, which are resistant to ccdB action. This was checked using ccdB resistant cell line available commercially from Invitrogen, USA (Cat # CI510-03). This indicated that the mechanism of action of LG is different than that observed with ccdB.
Genotype of ccdB resistant cell line
(F- mcrA D(mrr-hsdRMS-mcrBC) j801acZ DM15 DlacX74recAl ara D139 D(araleu)7697
galU galK rpsL (StrR ) endAI nupGtonA::Ptrc-ccdA)
The mechanism of action of LG molecule seems to be different from ccdB as evident from the killing of ccdB resistant cell line. Lethal effects of LG were studied in BL21 derived cell lines viz, BL21, HB101 (hybrid strain is isolated froman-E'.co//K12 x E.coli B cross), and found that, the LG was not active in both the cell lines and cells grew on plate containing LG inducer (Figure 5a and 5b) indicating that LG is lethal to only E. coli K12 derivatives and vs inactive in E. coli B strains.
To check whether the LG has any lethal effect on BL21 derived cell line in liquid medium, the BL21 Al cells were transformed with LG clone (pLUBT078) and induced with 13 mM L-Arabinose and 1% Lactose and absorbance was read at 600nm at every hour post induction for 3 hours. The absorbance of induced culture didn't drop (Table 3) supporting the observation that LG is not lethal to E. coli B strains. However, observations of lysis of BL21(DE3) cells upon LG expression indicates that the lysis is due to DE3 phage release and hence LG finds its applicability in prophage release which is a new and novel observation.
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Table 3: Absorbance of induced and Uninduced Culture at 600nm.

Hrs Uninduced (A600nm) Induced (A600nm)
0 0.81 0.8
1 1.33 0.87
2 2.16 0.92
3 2.3 0.84
4 2.4 0.7
5 2.5 0.72
6 2.88 0.74
7 3.4 0.67
Example 4A
Use of pBAD24m-LG (pLUBT112) as a cloning vehicle: Two genes namely hGCSF and Staphylokinase (SAK) gene were PCR amplified from synthetic DNA (Genscript, USA) and digested with EcoRI and ligated with EcoRl cut pLUBT112. DH5a cells were transformed with the ligation mix and plated onto LB-Amp and LBAmp-Arabinose plates. The plate without the insert showed very few (2-5) while the plate containing the insert showed several colonies. This indicated that every colony of the Vector+ insert plate must be a recombinant. A high efficiency of cloning (>95%) was observed when recombinants were screened by colony PCR and restriction digestion. Figure 6 shows the photographs of pLUBTl 12 carrying the genes of interest (GCSF and SAK gene) on plates with and without expression ofLG
Colonies from both the amp and amp-arabinose plates were selected for PCR screening of the GCSF and SAK clones. The results are shown in Figure 7 (a) & 7(b) and Figure 8(a) & 8(b).
It is clear from the figures that every colony picked up from the amp-arabinose plate showed the presence of required insert while only 1 of 10 colonies selected from the amp plate was a clone. Therefore the cloning efficiency was >95%. Similar results were obtained
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for SAK introduced into pLUBT112. The selected recombinants were crosschecked by restriction analysis to release the fragment of interest.
Example 4B
Non requirement of dephosphorylation of the LG vector
Usually any vector used for cloning purpose after restriction with single restriction enzymes require dephosphorylation using the commonly available calf intestinal phosphatase or Shrimp alkaline phosphatase, that are commercially available. This is done to reduce religation of the single cut vector by dephosphorylating one of the phosphate groups of one of the DNA strands. When such a vector is dephosphoralated and introduced into competent E.coli cells, there is approximately greater than 95% reduction in the number of colonies appearing as compared to non-dephosphorylated vector. There are some potential disadvantages with these enzymes. They are expensive and needs an additional time (nearly 2 hours) for performing this reaction.
In the present invention the vector described, pLUBT112, the LG vector does not require dephosphorylation at all, making it a very cost-effective and timesaving cloning vehicle.
On plates containing LG vector which is non-dephosphorylated but without the inducer, the number of colonies is nearly 100000 colonies per 100 ng DNA while the same vector with inducer shows only 0-5 colonies indicating >95% reduction in the number of colonies.
When different concentrations of inducers were used for checking the lethal activity of the LG vector, the plate without inducer contained nearly 1000000 colonies per) jag DNA while with the inducer (1, 10, 100 mM arabinose) the same vector showed only 0-10 surviving bacterial colonies, indicating>99% reduction in cell survival upon LG expression.
Figure 9 describes the photographs showing pLUBT112 without and with dephosphorylation. There is more than 99.99% reduction in colony number since LG is expressed in the panel B while it is not expressed in panel A.
Example 5
Effect of different amounts of inducer on LG lethality of E. coli DH5 alpha cells:
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pLUBT112 DNA was introduced into DH5 alpha cells and plated on plain LB amp plates containing different concentrations of arabinose namely 1, 10 and 100 mM to check if lower concentrations reduce the lethal properties of the LG gene. Results indicate that LG shows the lethal activity even at 1 mM concentration of arabinose (Figure 10)
Effect of different concentrations of the pLUBTl 12 DNA on lethal activity of the LG gene: Different concentrations of pLUBT112 was introduced into DH5 alpha cells and plated on 13 mM arabinose plates. Results indicate no survivors of the lethal action even when DNA concentrations are as low as 50 ng. (Figure 11)
Example 6
Effect of N terminal LG fusion constructs:
To ensure that the LG looses its lethal activity once linked with any foreign gene at the N terminus, the Lethal Gene is cloned at the C terminus of the GST gene (Glutathione S-transferase ) in pGEX4T-l vector. Results indicate that indeed LG looses its lethal activity when fused with foreign gene at the N terminus making it the most efficient tool for molecular cloning. LG was cloned in pGEX-4Tl at EcoRI and clones were screened by PCR and restriction digestion. DH5a cells were transformed with above clones and induced with ImM 1PTG. GST-LG fusion protein (-30 kDa) was seen insoluble fractions. (Figure 12)
The applications of the present invention includes an LG gene containing vector having following advantages as
(1) A dual potential vector that could be used both for cloning and expression studies.
(2) It requires arabinose as inducer, which is tightly controlled and hence does not require any special bacterial host for expression.
(3) It does not require dephosphorylation since single cut enzymes upon religation would die in presence of the inducer and double digested vector would not religate due to different overhangs.
(4) It gives essentially >95% recombinants as successful clones carrying the gene of interest. OI.
(5) It can be used successfully to clone DNA fragments of any size.
(6) The LG gene of the present invention could be used for lysis of the bacterial cells when introduced into a compatible plasm id/vector along with the plasmid carrying recombinant gene of interest. This is a cost effective process for bacterial lysis over the commonly used process
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in biotech industry where the bacterial lysis is usually performed with induced bacterial cell pellets using sonicator, french press, homogeniser.
(7) the only requirement of this vector ligation mix is it needs to be plated on antibiotic containing plates along with inducer that is arabinose (which is an inexpensive chemical).
(8) Since N terminal fusions of the LG did not show lethality, one could easily use this LG gene-containing vector for cloning experiments
(9) No antidote for this lethal gene is found yet and hence this would assure killing of strains completely.
(10) LG gene will be a potential agent to release prophages, this appears to be the first protein
which is a prophage inducing agent.
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SEQUENCE LISTING

<110>
<120>
<130> <140> <141> <150> <151> <160> <170>
<210> <211>
<212> <213>
<220> <221> <222> <223> <400>

: LUPIN LIMITED
:LETHAL GENE AS A TOOL FOR ZERO BACKGROUND
CLONING
: PCC3883
11
1
162 DNA
CDS
DNA sequence of the Lethal gene 1

atg ttt tat att ccc cag aac ate agg tta atg gcg ttt ttg atg tea ttt teg egg tgg ctg aga tea gcc act tct tec ccg ata acg gag ace ggc aca ctg gcc ata teg gtg gtc ate atg cgc cag ctt tea tec ccg ata tgc ace ace ggg taa

60
120
162

<210> <211> <212> <213> <220> <221> <222> <223> <400>
la
52 PRT
PEPTIDE
Amino acid sequence of Lethal gene la Met Phe Tyr He Pro Gin He Arg Leu Met Ala Phe Leu Met Ser Phe Ser Arg Trp Leu
15 10 15 20
Arg Ser Ala Thr Ser Ser Pro He Thr Glu Thr Gly Thr Leu Ala He Ser Val Val He
21 25 30 35 40
Met Arg Gin Leu Ser Ser Pro He Cys Thr Thr Gly Stop
41 45 50 52
19

<210> : 2
<211> : 633
<212> : DNA
<213>
<220>
<221> : miscfeature
<222> :
<223> : Nucleotide Sequence of ccdB Clone (in reverse
orientation)
<400> : 2
tgcggactct gtttctcata cccgtttttt tgggctagaa ataattttgt ttaactttaa 60
gaaggagata tacatatggc tagcatgact ggtggacagc aaatgggtcg cggatccgaa 120
ttcgagctcc gtcgacaagc tttttatatt ccccagaaca tcaggttaat ggcgtttttg 180
atgtcatttt cgcggtggct gagatcagcc acttcttccc cgataacgga gaccggcaca 240
ctggccatat cggtggtcat catcgccagc tttcatcccc gatatgcacc accgggtaaa 300
gttcacggga gacrttatct gacagcagac gtgcactggc cagggggatc accatccgtc 360
gcccgggcgt gtcaataata tcactctgta catccacaaa cagacgataa cggctctctc 420
ttttataggt gtaaacctta aactgcataa agcttggctg ttttggcgga tgagagaaga 480
ttttcagcct gatacagatt aaatcagaac gcagaagcgg tctgtaaaac agaatttgcc 540
tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 600
tagcgccgat ggtagtgtgg ggtctcccat gcg 633
<210> : 3
<211> : 556
<212> : DNA
<213>
<220>
<221> : misc_feature
<222> :
<223> : pET21a-LG clone
<400> : 3
atttatgagg acggaaaaaa ttccctctag aaatattttg tttactttag aggagatata 60
catatgtttt atattcccca gaacatcagg ttaatggcgt ttttgatgtc attttcgcgg 120
tggctgagat cagccacttc ttccccgata acggagaccg gcacactggc catatcggtg 180
gtcatcatgc gccagctttc atccccgata tgcaccaccg ggtaaaagct tgcggccgca 240
ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct 300
20

gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg 360
gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tggggacgcg 420
ccctgtacgg cgcattaagc gcggcgggtg tggtgtttac ccgcacgtga ccgctacaat 480
tgccacggcc taagcccgat ccattcaatt tcttcccttc cttttccccc aagttgccgg 540
ttttcccctt aaactt 556

<210> <211> <212>
<213> <220> <22J> <222> <223> <400>

4
588
DNA
misc feature
DNA sequence of the cloned junction pLUBT036 4

ctctgtttct ccatacccgt ttttttgggc tagaaataat tttgtttaac tttaagaagg 60
agatatacat atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga 120
gctccgtcga caagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga 180
ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg 240
tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg 300
tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag 360
tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg 420
acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg tggcgggcag 480
gacgcccgca taaactgcca ggcctcaaat taagcaaagg catcctgacg gatggctttt 540
tgggttctca actcctttgt ttttttcaaa aaattcaaag gttccgcc 588
<210> : 5
<211> : 776
<212> : DNA
<213>
<220>
<221> : misc_feature
<222> :
<223> : DNA sequence of pLUBTl 12
<400> : 5
21

tgttttctct gtttctccta cccgtttttt tgggctagaa ataattttgt ttaactttaa 60
gaaggagata tacatatggc tagcatgact ggtggacagc aaatgggtcg cggatccgaa 120
ttcgagctcc gtcgacaagc ttgcttttat attccccaga acatcaggtt aatggcgttt 180
ttgatgtcat tttcgcggtg gctgagatca gccacttctt ccccgataac ggagaccggc 240
acactggcca tatcggtggt catcatgcgc cagctttcat ccccgatatg caccaccggg 300
taaaagcttg gctgttttgg cggatgagag aagattttca gcctgataca gattaaatca 360
gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca 420
cctgacccca tgccgatctc agaagtgaaa cgccgtagcg ccgatggtag tgtgggggtc 480
tccccatgcc agagtaggga actgccaggc atccaaataa aacgaaaggg tcagtcgaaa 540
gactgggccc tttcgtttta tctgttgttt gtcggtgacg ctctcctgaa tgaggacaaa 600
tccggccggg agcgaatttg aacgttgcca agccacgacc ctaaggtggc cgcgagaacc 660
ccggtaataa cctgccggga tcaaattacc acagggcttc cccgacgatg tctctttttg 720
ttcacccccc ttttgttata tgtaaatttc aaataatccc ccggaagagt agccaa 776
<210> : 6
<211> : 45
<212> : DNA
<213>
<220>
<22l> : miscfeature
<222> :
<223> : forward primers for Lethal gene amplification by PCR
<400> : 6
ccg ccg gaa ttc cat atg ttt tat att ccc cag aac ate agg tta 45
<210> : 7
<211> : 45
<212> : DNA
<2I3>"
<220>
<221> : misc_feature
<222> :
<223> : Reverse primer for Lethal gene amplification by PCR
<400> : 7
ccg ccg gaa ttc aag ctt tta ccc ggt ggt gca tat egg gga tga 45
22

<210> <211> <212> <213>
<220> <221>
<222> <223>
vector
<400>
taa tac gac tca tag gg

20
DNA
misc_feature T7 forward and reverse primer for amplification from pET21a
20

<210> <211> <212>
<213> <220> <221> <222> <223>
9
19
DNA
: misc_feature
<400>
get agt tat tgc tea gq
<210> <211>
<212> <213>
<220> <22J> <222> <223>
: T7 forward and reverse primer for amplification from pET21a vectorT7Reverse primer : 9
19
10 39
DNA
misc feature
Forward Primer for Cloning of LG as Hindlll/Hindlll
fragment in 3pLUBT036
<400> : 10
ccg caa get tgc ttt tat art ccc cag aac ate agg tta 39
23

<210> : 10
<211> : 45
<212> : DNA
<213>
<220>
<221 > : misc_feature
<222> :
<223> : Reverse Primer for Cloning of LG as Hindlll/Hindlll fragment
in pLUBT036
<400> : - 11
ccg ccg gaa ttc aag ctt tta ccc ggt ggt gca tat egg gga tga 45
24