A genetic tool for engineering the genome of bacteria and a method of preparing the same there of

Abstract

The present invention relates to the field of Biotechnology and genetic engineering. In particular, the present invention relates to the field of a novel genetic tool with site-specific integration and FLP-FRT recombination excision of antibiotic selection marker for utility in applied and industrial research, biotherapeutic development and diagnostics.

Description

[0001]B5105-00319 A GENETIC TOOL FOR ENGINEERING THE GENOME OF BACTERIA AND A METHOD OF PREPARING THE SAME THERE OF FIELD OF THE INVENTION: The present invention relates to the field of Biotechnology and genetic engineering. In particular, the present invention relates to the field of a novel genetic tool with site- specific integration and FLP-FRT recombination excision of antibiotic selection marker for utility in applied and industrial research, biotherapeutic development and diagnostics. BACKGROUND OF THE INVENTION: The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. An expression vector is also known as expression construct. Mostly, it is a plasmid or virus structurally modified for gene expression in cells. A vector is a biological vehicle carrying a specific gene of interest that can be incorporated into a target cell that can operate to produce specific protein. Expression vectors are the basic tools in biotechnology for the production of proteins. Integrative expression vector can be designed to introduce single or multiple gene of interest into bacterial chromosome for biotherapeutic, vaccine research and metabolic engineering of bacteria with novel phenotypes for environmental applications. Further refinement in selecting a suitable promoter capable of facilitating either constitutive or inducible expression of gene of interest enables precise manipulation of gene expression tailored to specific applications. The FLP-FRT system and Cre-lox systems are highly effective and extensively employed site-specific recombinase technology in genetic engineering. Through strategic positioning of two FRT (locus of crossover) sites, FLP (recombinase) proteins proficiently execute DNA deletion, insertions, translocations and inversions. The FRT is a 48 base pair (bp) sequence that is incorporated at both sides flanking the gene of B5105-00319 interest that has to be deleted. When the recombinase is expressed transiently, the gene- flanked site directly by the FRT sequence is excised from the genome by homologous recombination mediated by the recombinase. The Cre / FLP recombinases are tyrosine family site-specific recombinases that utilise type IB topoisomerase mechanism causing the recombination of two separate strands of DNA. The prior art discloses certain integrative vectors, processes for preparing these integrative vectors, vectors with FLP-FRT recombination activity and removal of antibiotic resistance marker. Some of the literature is as mentioned below: 1. A Novel, Broad-Range, CTX-Derived Stable Integrative Expression Vector for Functional Studies (doi: 10.1128 / JB.01966-14) – explains the construction of an integrative expression vector that utilizes XerC and XerD recombinases to irreversibly integrate into the dif loci of the host chromosome. The expression vectors pBD62 and pBD66 have an arabinose inducible promoter, XBS of CTXΦ which facilitates the integration and a zeocin selectable marker. 2. Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker (doi: 10.1093 / nar / gkr1321)– explains the development of an integrative vector that utilises the allele coupled exchange (ACE) strategy to select double crossover clones without using a plasmid-borne counter selectable marker. In the strategy, the double cross-over clones were selected on erythromycin as they have ermB allele. 3. A rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies (doi.10.1038 / srep09684) – explains a strategy for chromosomal integration of gene (s) with multiple copies utilizing the flippase from the yeast 2 μm plasmid. 4. Fast and antibiotic free genome integration into Escherichia coli chromosome (doi.org / 10.1038 / s41598-020-73348-x) – explains the development of an integration strategy into E.coli genome combining λ-red recombination system with the site-directed homing endonuclease I from Saccharaomyces cerevisiae (I-SceI) for selection. B5105-00319 5. Application of FLP-FRT system to construct unmarked deletion in Helicobacter pylori and functional study of gene hp0788 in pathogenesis (doi.org / 10.3389 / fmicb.2017.02357) – explains the construction of a FLP expressing plasmid pCHF for unmarked deletion of genes from H. pylori genome to study its functionality. The transformants were selected on kanamycin plates. 6. Plasmid Vectors for in Vivo Selection-Free Use with the Probiotic E. coli Nissle 1917 (doi.org / 10.1021 / acssynbio.0c00466)-explains the development of pMUT plasmids for genetic engineering of E.coli Nissle 1917 strain alongside a CRISPR-Cas9 system to remove the endogenous plasmid. 7. Application of the FLP / FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae (doi: 10.1002 / yea.1895) – explains the use of Flp / FRT system for conditionally delete genes from the genome of yeast Saccharomyces cerevisiae. 8. A broad-host-range FLP-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants (doi.org / 10.1016 / S0378- 1119(98)00130-9) – explains the utilization of FLP-FRT system for site-specific excision of chromosomally located DNA sequences. The paper explains the methodology of the construction of a new GmR-FRT cassette and a broad-host- range (bhr) FLP recombinase-expressing vector. However, the above-mentioned prior art and the other known documents suffer with the following limitations: 1. In most existing systems, the stability of chromosomal integration is a major cause of concern as chromosomal integration in different loci has different stability. 2. Most of the vectors employ antibiotic selection markers to ensure robust expression and stability in the host cells facilitating its integration alongside the gene of interest in the host chromosome. An essential criterion in biotherapeutic and vaccine-producing strain development is ensuring that the strain is free of B5105-00319 any additional exogenous antibiotic resistance cassette, regardless of its transferability status. 3. Most of the integrative expression vectors have limited host range restricting their utility to be employed for genetic manipulation of multiple hosts. Therefore, to overcome the limitations associated with the vectors in prior art, there is a need for the development of an irreversibly integrating expression vector that can ensure stable expression of the gene of interest. Moreover, it must be amenable to manipulate to yield a final strain devoid of antibiotic selection, thereby ensuring versatile applicability. OBJECTIVE OF THE INVENTION: The primary object of the present invention is to overcome the drawbacks associated with prior art. Another object of the present invention is to provide a novel genetic tool with site- specific integration and FLP-FRT recombination excision of antibiotic selection marker for utility in applied and industrial research, biotherapeutic development and diagnostics. Another object of the present invention is to provide an expression vector which can be irreversibly integrated thus ensuring stable expression of the gene of interest. Another object of the present invention is to provide integrative expression vectors which can be used in a variety of host ranges without any restriction, for genetic manipulation of multiple hosts. Another object of the present invention is to provide a method of producing the expression vector which can be irreversibly integrated thus ensuring stable expression of the gene of interest. SUMMARY OF THE INVENTION: The Invention provides an integrative expression vector construct comprising: a) pBD62 parent vector comprising: B5105-00319 i) XerC and XerD binding sites (XBS) amplified from the CTXΦ; ii) a glycopeptide antibiotic Zeocin neutralizing gene sh-ble cloned from V. cholerae isolate IDH06781 genome; iii) transcriptional regulator AraC, encoded by the araC cloned from pBAD24 along with PBAD promoter segment; and iv) a DNA segment containing binding sites for multiple restriction enzymes (MCS); b) a constitutive promoter comprising htpG in pBD62 vector, that allows continuous expression of any gene of interest cloned under it, positioned after the AraC promoter; c) a sh-ble gene flanked by 48bp FRT sequences (FRT-sh-ble-FRT), which allows conditional deletion of the antibiotic resistance gene, into pSB49; d) atleast a DNA segment comprising binding sites for multiple restriction enzymes, positioned after the htpG promoter by restriction digestion and ligation, to improve the MCS; e) said constitutive promoter htpG is preceded by a ctxB secretory signal peptide for the secretion of the gene of interest followed by a useful multiple cloning site. The Invention provides a method of constructing the integrative expression vector construct as described above, comprises the steps of: a) inserting a constitutive promoter comprising htpG in pBD62 vector, that allows continuous expression of any gene of interest cloned under it, after the AraC promoter; b) cloning a sh-ble gene flanked by 48bp FRT sequences (FRT-sh-ble-FRT), which allows conditional deletion of the antibiotic resistance gene, into pSB49; and amplifying the FRT-sh-ble-FRT sequence from pEE23 vector; c) introducing a DNA segment comprising binding sites for multiple restriction enzymes, after the htpG promoter by restriction digestion and ligation, to improve the MCS; B5105-00319 d) cloning a ctxB signal peptide sequence of 72 nucleotides into pDL31 for the secretion of the gene of interest encoded protein outside the host bacterial cells. BRIEF DESCRIPTION OF DRAWINGS: To further clarify the advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which are illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting in their scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings in which: Figure 1 illustrates the ‘vector diagram of pBD100’ and ‘construction strategy of pBD100. The final construct, pBD100, was confirmed by Sanger sequencing. DETAILED DESCRIPTION: For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof. The invention provides an irreversibly integrating expression vector which can ensure stable expression of the gene of interest which could be amenable to manipulate to yield a final strain devoid of antibiotic selection cassette for biotherapeutic, vaccine and other utility in applied and industrial research, diagnostics and manufacturing. B5105-00319 In an embodiment, the invention discloses, a novel indigenous expression vector by recombining traits from bacterial chromosomes, plasmids and phage genomes. The present invention also discloses a method for constructing the vector. The vector possesses a replicative and / or integrative form with potential stable transcription of the gene of interest. Additionally, the vector has a signal peptide sequence after the promoter and before the multiple cloning site which could be utilized to ensure secretion of the protein of interest out of the bacteria. Moreover, the strategy utilizes the FLP- FRT system to manipulate the vector to yield a final strain devoid of antibiotic selection as per its applicability. Furthermore, the vector ensures applicability in a broad host range. The vector, of the present invention, is a useful genetic tool for the expression of potential biotherapeutic molecules and antigens from different microbes in the interested bacterial species. This could accelerate drug discovery research by facilitating rapid evaluation of gene functions. The novel designed vector, of the present invention referred herein as pBD100, is depicted at Figure 1 and incorporated herein in its entirety. The integrative vector of the present invention exploits part of the CTX^ genome, part of the conditionally integrative vector pBD62 and a FLP-FRT recombination system. The vector possesses a constitutive promoter, htpG which ensures stable expression of the gene of interest, which is preceded by a ctxB secretory signal peptide sequence followed by a useful multiple cloning site. The vector of the present invention irreversibly integrates into the dif loci present in many Gram-negative bacterial hosts. The present invention utilises the same method as of pBD62 for the stable site-specific irreversible integration of the vector into the dimer resolution site (dif) of the host chromosome. In particular, the present invention utilises the unique mode of integration of CTX^ into the bacterial chromosome exploiting tyrosine recombinases XerC and XerD, as such integration is highly efficient and irreversible. The vector of the present invention includes a constitutive promoter, htpG, strong ribosomal binding site (RBS), XerC and XerD binding sites (XBS) and attP1 and attP2. During integration, the 150- bp region of ig-1 encompassing the attP1 and attP2 forms intra-strand base-pairing interactions and generates a functional phage attachment site, attP(+). The attP(+) of CTX^ is recognized by the host-encoded XerC-XerD enzymes and helps in integrating B5105-00319 at the dif site by site-specific recombination. The XerC and XerD binding sites of the vector are compatible with the chromosome dimer resolution system of several bacterial species such as Vibrio cholerae, V. fluvialis, V. parahaemolyticus, Escherichia coli, Klebsiella pneumoniae, Salmonella enterica and others and thus the host range of the present vector is very large. In an embodiment, the present invention discloses the method of constructing the vector pBD100 of the present invention wherein different steps, as mentioned below, are used: • pBD62 (doi: 10.1128 / JB.01966-14) was used as the parent vector which possessed the XerC and XerD binding sites (XBS) amplified from the CTXΦ. The pBD62 vector additionally has a glycopeptide antibiotic Zeocin neutralizing gene sh-ble cloned from V. cholerae isolate IDH06781 genome, transcriptional regulator AraC, encoded by the araC cloned from pBAD24 along with PBAD promoter segment, and a DNA segment containing binding sites for multiple restriction enzymes (MCS) • To the pBD62 vector, a constitutive promoter, htpG that allows continuous expression of any gene of interest cloned under it was inserted after the AraC promoter. The htpG was amplified from the V. cholerae genome and cloned into pBD62 using the restriction enzymes PstI and SpeI (primers 1195F and 1196R) to construct pSB49. • Further, a sh-ble gene flanked by 48bp FRT sequences (FRT-sh-ble-FRT) which allows conditional deletion of the antibiotic resistance gene was cloned into pSB49. The FRT-sh-ble-FRT sequence was amplified from the pEE23 vector. PCR amplification, restriction digestion and DNA ligation techniques were used for cloning both the elements. The primers used were 1817F and 1818R and the restriction enzymes used were NdeI and ScaI. The construct was named pDL30. • Next, to improve the MCS, a DNA segment containing binding sites for multiple restriction enzymes, was introduced after the htpG promoter by restriction digestion and ligation. The vector was named pDL31. B5105-00319 • Further, a ctxB signal peptide sequence of 72 nucleotides was cloned into pDL31 allows secretion of the gene of interest encoded protein outside the host bacterial cells. Thus, the integrative expression vector pBD100 carries a conditional replication origin ori R6K, FRT-sh-ble-FRT sequence that allows conditional excision of the antibiotic selection marker, constitutive promoter htpG, inducible AraC promoter, secretory signal sequence, conjugative transfer sequence RP4 and binding sites for multiple restriction enzymes (MCS). In another embodiment, the vector pBD100 may be utilized as a suitable genetic tool for genome engineering and functional genomics for most bacterial species making it widely applicable across many bacterial taxa. Also, pBD100 may be utilised to develop bacterial strains as potential biotherapeutic agents, vaccine candidates, metabolically advanced for environmental and industrial application by introducing specific gene(s) of interest. The novel integrative vector with conditional antibiotic selection gene excision of the present invention may be considered as a suitable genetic tool to deliver single or multiple function encoding genes in the chromosome of interested bacterial species which may help us to develop environmentally safe and effective biotherapeutic isolates, live attenuated or killed whole cell bacterial vaccines and metabolically enhanced bacterial strains for industrial and agricultural applications. Since, the chromosomal integration site of the current vector is highly conserved in bacterial phyla, the host range of the vector is very wide. Without being limited by theory, the present invention of the integrative expression vector with conditional antibiotic selection marker deletion / excision ability integrates at the dimer resolution site in bacterial chromosome and replicates passively with host chromosomal replication. The antibiotic resistance cassette in the vector could be deleted within the host bacterial cell by the transient expression of another thermolabile vector encoding the Flp recombinase. The temperature-sensitive vector encoding the recombinase can be conjugally transferred into the strain and can be cured off by incubating at a high temperature of 42ºC. The vector of the present invention does not require any selection pressure for its stable maintenance. Due to its integrative nature, it is possible to maintain the copy number accurately in the host cells and as the B5105-00319 antibiotic selection marker is flanked with FRT cassette, it can be deleted conditionally after confirming the positive clones. It has a constitutive promoter which allows constitutive expression of the gene of interest cloned under it, ribosomal binding site, wide range of selection opportunity, and a broad host range. The integrative module, XerC and XerD binding site, is compatible with multiple bacterial species and could be useful for precise expression of recombinant proteins in multiple bacterial cells including E. coli, V. cholerae, V. fluvialis, V. parahaemolyticus, Shigella dysenteriae, S. enterica, K. pneumonia and Aeromonas hydrophila. Advantages • The vector of the present invention contains part of the bacteriophage CTX^ genome that provides XerC-XerD binding site for irreversible integration at the dimer resolution site (dif) in the bacterial chromosome. • The vector of the present invention contains stable constitutive promoters for continuous robust expression of the gene of interest. • The vector of the present invention contains zeocin antibiotic resistance selection marker for easy selection of transconjugants. • In the vector of the present invention, the zeocin resistance encoding sh-ble gene is flanked by FRT sequences that are recognised by tyrosine recombinases which allows conditional deletion of the resistance gene from the transconjugants to generate a final strain devoid of any additional resistance alleles. • Additionally, the vector has a signal peptide sequence after the promoter and before the multiple cloning site which could be utilized to ensure secretion of the protein of interest out of the host bacterial cell.

Claims

B5105-00319 We Claim:

1. An integrative expression vector construct comprising: a) pBD62 parent vector comprising: i) XerC and XerD binding sites (XBS) amplified from the CTXΦ; ii) a glycopeptide antibiotic Zeocin neutralizing gene sh-ble cloned from V. cholerae isolate IDH06781 genome; iii) transcriptional regulator AraC, encoded by the araC cloned from pBAD24 along with PBAD promoter segment; and iv) a DNA segment containing binding sites for multiple restriction enzymes (MCS); b) a constitutive promoter htpG obtained from Vibrio cholerae genome, that allows continuous expression of any gene of interest cloned under it, positioned after the AraC promoter; c) a sh-ble gene flanked by 48-bp FRT sequences (FRT-sh-ble-FRT), which allows conditional deletion of the antibiotic resistance gene sh-ble, from the vector after its integration in the chromosome; d) at least a DNA segment comprising binding sites for multiple restriction enzymes, positioned after the htpG promoter by restriction digestion and ligation, to improve the MCS; e) said constitutive promoter htpG is preceded by ribosomal binding site (RBS) and a protein secretory signal peptide from ctxAB for the secretion of the peptide of interest.

2. The construct as claimed in claim 1, wherein XerC and XerD binding sites of the vector are compatible with the chromosome dimer resolution system of several bacterial species comprising Vibrio cholerae, V. fluvialis, V. parahaemolyticus, Escherichia coli, Klebsiella pneumoniae, Salmonella enterica and others and thus the host range of the present vector is very large. 1B5105-00319 3. The construct as claimed in claim 1, wherein attP(+) of CTX^ is recognized by the host-encoded XerC-XerD enzymes and helps in integrating at the dif site by site-specific recombination.

4. The construct as claimed in claim 1, wherein the vector irreversibly integrates into the dif loci present in many Gram-negative bacterial hosts.

5. The construct as claimed in claim 1, comprises the nucleotide sequence represented by sequence Id 1. (The Inventors are requested to provide the nucleotide sequence) 6. A method of constructing the integrative expression vector construct as claimed in claim 1, comprising the steps of: a) inserting a constitutive promoter comprising htpG in pBD62 vector, that allows continuous expression of any gene of interest cloned under it, after the AraC promoter; b) cloning a sh-ble gene flanked by 48bp FRT sequences (FRT-sh-ble-FRT), which allows conditional deletion of the antibiotic resistance gene, into pSB49; and amplifying the FRT-sh-ble-FRT sequence from pEE23 vector; c) introducing a DNA segment comprising binding sites for multiple restriction enzymes, after the htpG promoter by restriction digestion and ligation, to improve the MCS; d) cloning a ctxB signal peptide sequence of 72 nucleotides into pDL31 for the secretion of the gene of interest encoded protein outside the host bacterial cells.

7. The method as claimed in claim 6, wherein during integration, the 150-bp region of ig-1 encompassing attP1 and attP2 forms intra-strand base-pairing interactions and generates a functional phage attachment site, attP(+).

8. The method as claimed in claim 6, wherein the htpG is amplified from the V. cholerae genome and cloned into pBD62 by the restriction enzymes comprising PstI and SpeI from the primers 1195F and 1196R, to construct pSB49. 2B5105-00319 9. The method as claimed in claim 6, wherein amplifying the FRT-sh-ble-FRT sequence from pEE23 vector by primers comprises 1817F and 1818R and the restriction enzymes comprises NdeI and ScaI. 3

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