DESC:FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patent Rule, 2003
COMPLETE SPECIFICATION
(see section 10 and rule 13)

TITLE OF INVENTION
Platform technology for Antibiotic-free process for production of biotherapeutics

Applicant(s)
AMELIORATE BIOTECH PRIVATE LIMITED
NO.1213, BCCS LAYOUT HOLIDAY VILLAGE ROAD, VAJARAHALLI KANAKAPURA MAIN ROAD, BANGALORE-560062, Karnataka, India

The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION:
The present invention relates to expression and production of bio-therapeutics using a unique cloning platform technology for antibiotic free process.
The present invention particularly relates to clone the PtsI gene of the E.coli cell under a constitutive promoter in a high copy number recombinant vector.
BACKGROUND OF INVENTION:
The use of antibiotics for therapeutic application against some infectious agents has been practiced for several decades. The consequences include emergence of resistant or even multi-resistant pathogenic bacterial strains.
Antibiotics are often used as a selection pressure to avoid bio-contamination and to pre-vent plasmid loss in production processes such as fermentation. That could be used as the antibiotic molecule itself in a given biological product and the antibiotic resistance gene used as a selection marker.
Health agencies and global organizations (FDA, EMEA, WHO) are all going in the same di-rection concerning recommendations towards the limitation in use of antibiotics and propagation of antibiotics resistance genes. The rationale for the use of antibiotics must be clearly documented in the Common Technical Document (CTD) since it’s strongly ad-vised to avoid or minimize the use of any kind of antibiotics in cell or bacterial culture. The presence of a gene for resistance to an antibiotic is now considered a major draw-back for use in humans.
The regulatory requirements on biological agents are increasing day by day, which may have a great impact in the field of industrial protein expression and production, live vec-tors for bio-therapeutic agent delivery and gene therapy. It is expected that in the near future, the rule becomes “zero tolerance” towards antibiotic-based selection in produc-tion and delivery systems. The complete absence of antibiotic-resistance genes being the only way to ensure that propagation in the environment or transfer of resistance to pathogenic strains will not happen.
The use of the ß-lactam antibiotic family is not allowed to avoid cases of sensitivity. Peni-cillin, and more generally ß-lactams and streptomycin, must not be used in reason of po-tential concerns with hyper reactivity of some patients to antibiotics of the ß-lactam family. Kanamycin and Tetracycline are commonly used and are still acceptable to health authorities.
Antibiotic-resistant bacteria are causing a global health crisis because of the horizontal genetic transfer of antibiotic resistant genes to prokaryotic organisms present in the envi-ronment or in commensal flora. Horizontal genetic transfer (HGT) is the passage of genet-ic elements between organisms. HGT is influenced by the advantage conferred by the transferred gene, the toxicity of its product, the capacity of the transferred gene to be integrated into the host genome and to be stabilized, and a certain compatibility of co-don usage between the transferred gene and the host. The probability of integration and stabilization of a HGT into a new host genome is correlated and increased by the similari-ty between the tRNA pools of the donor and recipient organism. Acquisition of antibiotic resistance is one example of this evolution by HGT.
In 1965, Datta and Kontomichalou showed the widespread transfer of penicillin re-sistance across Enterobacteriaceae. A marked ß-lactamase activity was also measured in various Pseudomonas aeruginosa resistant strains in the early seventies. More recently, acquisition of the virulence factors that distinguish Salmonella sp. from Escherichia coli has been clearly shown as the result of horizontal gene transfer. Recent technological breakthroughs, such as the one that can be observed for next generation sequencing, of-fers unthinkable and extensive possibilities of detecting numerous HGT events, especially if the data acquisition is completed by new bioinformatics tools like nucleotide substitu-tion rate matrixes. Moreover, if we consider the situation at the microbiome level, it be-comes obvious that a multitude of different bacterial species, exhibiting many types of complex interactions, are likely to use HGT as a preponderant mechanism for adaptation in difficult environment.
Plasmid instability is another significant concern in the academic and industrial utiliza-tion of microorganisms for protein or DNA production. Usually, these processes require the use of a bacterial plasmid construct as a vector carrying a gene to be expressed. It has been demonstrated that the growth rate of plasmid-bearing cells is significantly reduced relative to that of a plasmid-free cells, simply because plasmid replication and transcrip-tion, as well as protein production, represent a significant burden on cellular metabo-lism. Hence, in a fermentation process, cells losing the plasmid construct exhibit a higher fitness than construct-free cells and the former rapidly overcome the latter in the bacte-rial population. Antibiotic resistance genes are the most commonly used selectable markers in fermentation procedures to avoid plasmid free cells to survive and dominate the culture.
The presence of an antibiotic resistant gene in the vector backbone for specific therapeu-tic agents or fields of application such as DNA vaccination, passive immunization using antibody gene transfer or gene therapy, is a major concern mainly because of possibility of horizontal transfer of antibiotic resistance to circulating microbial population. Such a consideration is emphasized by the very long persistence of DNA constructs upon injec-tion.
Cranenburgh, R.M. et al. reported the construction of two novel Escherichia coli strains (DH1/lacdapD and DH1lacP2dapD) that facilitate the antibiotic-free selection and stable maintenance of recombinant plasmids in complex media. They contain the essential chromosomal gene, dapD, under the control of the lac operator/promoter (Cranenburgh, R. M., et al., 2001). Unless supplemented with IPTG (which induces expression of dapD) or DAP, these cells lyse, however, when the strains are transformed with a multicopy plas-mid containing the lac operator, the operator competitively titrates the LacI repressor and allows expression of dapD from the lac promoter. Thus transformants can be isolat-ed and propagated simply by their ability to grow on any medium by repressor titration selection. No antibiotic resistance genes or other protein expressing sequences are re-quired on the plasmid, and antibiotics are not necessary for plasmid selection.
The European patent 2310511 provides a self-replicative vector lacking the resistance gene to an antibiotic comprising a coding sequence operably linked ccdA protein to a first promoter, the sequence of the Cer locus, a heterologous sequence operably linked to a second promoter. The heterologous sequence encodes the rEPA protein.
The European patent 2432884 provides an engineered gram-negative bacterium host strain comprising a drugless plasmid wherein said drugless plasmid comprises a polynu-cleotide encoding a cI repressor protein. Methods disclosed include methods of produc-ing antibiotic-free plasmids and methods for transfer of foreign genes into mammalian cells using the antibiotic-free plasmids wherein the heterologous polynucleotide com-prises a sacB gene which encodes a SacB protein.
The U.S. patent 7736899 mentions the construction of an E. coli strain including a Cys-+Val missense mutation at the thymidylate synthase active site and creating a nutritional requirement for thymine, thymidine or cysteine. The artificial alleles of the thyA gene are constructed by site-directed mutagenesis of the plasmid pTS0 (Lemeignan et al. 1993), which derives from the plasmid pTZ18R (BioRad) by insertion of the wild-type thyA gene of E. coli. The plasmid pTS1 thus obtained propagates the thyA:Val146 allele, in which po-sition 146 occupied in the wild-type thyA gene by the UGC codon of cysteine is occupied by the GUA codon of valine. The plasmid pTS1 is introduced by transformation, carried out according to the method of Sambrook et al. (1989), into the E. coli K12 ?thyA strain, ß1308 (Lemeignan et al., 1993), in which the chromosomal thymidylate synthase gene, thyA, is deleted.
Antibiotic-free recombinant plasmid selection and stabilization in E.coli based on the auxotrophy complementation of the activity of TpiA has been demonstrated (Ram Shan-kar Velur Selvamani et al, 2014). They were able to achieve antibiotic-free cloning, selec-tion, expression of a model recombinant product and long-term stability of the plasmid in continuous culture. The growth advantage shown by the plasmid-complemented strain even under non-selective conditions makes the system particularly attractive for large-scale industrial processes.
The Phosphoenolpyruvate Sugar Phosphotransferase System (PTS) is a multicomponent system and catalyzes vectorial phosphorylation of various sugars. Thus, in E. coli, for ex-ample, glucose and other sugars are translocated across the membrane by PTS-catalyzed phosphorylation (Lan Guan et al, 2004). By regulation of transcription of lacY, as well as LacY activity by IIAGlc of the PTS, E. coli grown in the presence of a mixture of glucose and lactose utilize the lactose only when the glucose in the medium is completely exhausted, leading to diauxic or biphasic growth.
In brief, the problems with current state of art are mentioned below:
1. Antibiotic usage leading to increase environmental hazard (development of anti-biotic resistant strains),
2. Low yield (due to plasmid stabilisation issue).
3. Higher cost (assessment of residual antibiotic levels and, if required, their remov-al), which in turn affects drug affordability to the Indian population.
In past, several alternatives have been proposed.The antibiotic use related issue can be addressed by using alternative selection pressure, i.e. Gene knock down strategy or bactericidal expression.
In house-keeping gene knock down strategy, the House-keeping gene is introduced in the plasmid. This gene is knocked down from the host genome. Normally, in a cell, one or two copies of house keeping gene is required but since this gene was cloned in the plasmid,wherein the plasmid copy number was 50 to 100 or even more, it led to the pro-duction of that particular house-keeping gene in excess. This situation is not desirable since it will be toxic for the cell. In this situation, the cell might lose the plasmid in an at-tempt to survive, or cell death can happen.
The approach of Bactericidal expression is similar to the introduction of antibiotic re-sistance gene as selection marker. In this method, instead of antibiotic resistance gene, some gene coding for enzyme is introduced. The enzyme breaks down the toxin present in the media and hence selection of recombinant clone can be done. This technology had the same disadvantages as antibiotic resistance based selection strategy as it leads to plasmid loss and also had scale-up and yield issue.
All these strategies do not address the plasmid stabilisation issue which results in low yield. So the present invention addresses those issues in such a way that no antibiotic is used in the process without compromising yield. The aim of present invention is to ad-dress these issues by different and complementary approaches. Our strategy is to substi-tute the antibiotic-based selection by an alternative mean such as the complementation of an essential gene product, not expressed by the host, poison antidote mean of selec-tion or sophisticated post-segregation killing mechanism.
SUMMARY OF INVENTION
The present invention descrbes a process of selective nutritional pressure for producing a recombinant protein, wherein the process comprises of following steps:
A. knocking out of gene from host cell by process of homologous recombination
B. transforming host cell with a vector comprising a gene encoding the recombi-nant protein and knocked-out gene placed under a constitutive promoter
C. Growing the transformed host cells of step B in minimal media
D. Obtaining recombinant protein expressed.
OBJECT OF INVENTION
The object of the invention is to provide a novel cloning platform technology by nutri-tional selection procedure, leading to production of bio-therapeutics, by a completely antibiotic free process.
More particularly, the object of the present invention is to provide a system to propagate recombinant E.coli strain in absence of antibiotic selection by model of nutritional selection pressure wherein it is complimented by a specific vector carrying the required gene.
BRIEF DESCRIPTION OF DRAWINGS:
The invention will be described in detail in the following description with reference to the figures in which:
Figure 1 is a schematic representation of Glucose transportation inhibition in mutant strain of E.coli.
Figure 2 is a schematic representation of selection of E. coli transformants (of knockout Pts1 gene) on M9 + Glucose media.
Figure 3 is a schematic representation of Map of pRed/ET plasmid used for the transfor-mation of host cell E. coli
Figure 4 is a schematic representation of pKD13 vector for host cell E. Coli transformation with its Kan/Neo cassette
Figure 5 is a representation ofthe colonies after transformation of host cell with pRed/ET plasmid on LA-Amp plate
Figure 6 is a representation of induced and un-induced plates after electroporation with linear Kan/Neo cassette in pRed/ET transformed dH5 alpha cells
Figure 7 is a representation of colony PCR with insert specific primers designed on Kan/Neo cassette on colonies screened for pts1 gene knockout. Plasmid pKD-13 was used as positive control for amplification of DNA from Kan/Neo cassette.
Figure 8 is a representation of PCT amplification on the genomic DNA isolated from re-combinant strain and wild type strain. NTC is ‘no template DNA control’ confirming the template dependent amplification in PCR positive lanes.
Figure 9 is a representation of results when pts1 knockout strain was subjected to nutri-tional selection in M9 minimal media supplemented with Glucose as well as in Luria Ber-tani Agar. Both the plates have Kanamycin selection. Pts1 mutants failed to grow on M9 media as they failed to utilize Glucose as a sole source of carbon. A recombinant DH5a strain transformed with a pET28b* plasmid was used as a control.
Figure 10 is a representation of digestion of final consutrct of Ranibizumab with Pts1/Clal for PST1 dropdown (approx. 1.7kb) and Sac/Sall for Rbz dropdown (approx. 1.5kb).
DESCRIPTION OF INVENTION
Definitions
The terms "protein", "peptide", "polypeptide" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid pol-ymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipula-tion or modification, such as conjugation with a labeling or bioactive component.
The term "gene" is used broadly to refer to segment of polynucleotide associated with a biological function. Thus, genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their ex-pression. For example, gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequenc-es.
A "host cell" denotes a prokaryotic or eukaryotic cell that has been genetically altered or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. When referring to genetically altered cells, the term refers both to the originally altered cell and to the progeny thereof.
The term “M9 media” is used to refer ATCC Medium: 2511 M9 Minimal Agar/Broth which is a solid media containing M9 salts (such as Na2HPO4:12.8g, KH2PO4:3.0g, NaCl:0.5g, NH4Cl:1.0g DI Water:478 ml) and Agar colution (such as Agar:15g, DI water: 500ml). Upon Autoclaving Glucose:20% Solution:20 ml, 1M MgSO4 Solution:2ml, 1M CaCl2 Solution:0.1ml, Thiamine (0.5% w/v Solution):0.1 ml is further added.
The term “culturing” “propagation” “growth” are used and referred to mean increase of microbial organisms in numbers by letting them reproduce in predetermined culture me-dium under controlled laboratory conditions.
The term “recombinant protein includes but not limited to granulocyte colony stimulat-ing factor (Gcsf), interferon, parathyroid hormone, interleukin, reteplase, thrombolytic agent, antibody, insulin and its analogues.
The present invention aims to provide a new process used at industrial scale that has the double advantage of resulting in a high expression yield and in the absence of any use of antibiotic and can therefore be scaled up to large volumes.
According to the present invention referred herein as Nutritional selection, ptsI gene, which is involved in glucose transport, was knocked out from host, by the process of ho-mologous recombination. This gene was introduced in the plasmid. However, in contrast to housekeeping gene selection strategy, in this case, the gene product does not affect the cell viability and growth. Even if media contains minimum amount of glucose, still plasmid produces ptsI gene product, in an attempt to find glucose in media. It becomes a necessity for the cell to retain the plasmid and continue growth, because it needs glucose to survive (table 1 describes the advantages of present invention).
Parameters Conventional process Present Invention
Clone Antibiotic selection marker No antibiotic
Plasmid stability Loss of plasmid in certain sit-uations Stable plasmid
Yield low yield,
batch to batch variation ob-served high yield,
no batch to batch variation ob-served
Scalability Not easy Easy
Regulatory approval Difficult Easier
Analysis cost High Low
Overall process time High Low
Overall cost of prod-uct High Low

Table1: Comparison of conventional process with the process of present invention

In particular, the present invention comprises of following steps:
1. An E.coli strain such as K-12, genetically modified, deficient in glucose metabolism in post transportation phosphorylation cascade pathway (pts1 gene)is developed.
2. Specific vector carrying the required gene (pts1 gene). A polycistronic gene encoding Ranibizumab antibody heavy and light chain is cloned in the vector.
3. Culturing both host cell and vector in the medium containing only glucose as carbon source.
4. Only the cells, harbouring the vector (plasmid) survive in a minimal media containing glucose.
5. Selected colonies synthesize the over-expressed Ranibizumab in an antibiotic free media.

Host Cell (Escherichia coli) Modification
Escherichia coli K12 strain was considered as a host cell for the recombinant protein expression for the present invention.
Glucose transport to the E.coli cell and its metabolism is regulated by a cascade pathway. The Glucose enters the cell through the cell membrane. Unless and until Glucose gets phosphorylated, it remains permeable through the cell membrane. The phosphate group is transferred to Glucose from PhosphoenolPyruvate through a cascade pathway. The first enzyme involved in this pathway is encoded by a gene named Pts1 (Phospho Transferase System, Enzyme 1) (Figure 1).

In many bacteria, the phosphoenolpyruvate:glycose phosphotransferase system (PTS) is involved in the uptake and concomitant phosphorylation of a variety of carbohydrates. The PTS is a group transfer pathway; a phosphoryl group derived from phosphoenolpy-ruvate (PEP) is transferred sequentially along a series of proteins to the carbohydrate molecule. The sequence of phosphotransfer is from PEP to the general cytoplasmic PTS proteins enzyme I (EI) and HPr and, in the case of glucose, further to the carbohydrate-specific cytoplasmic IIAGlc, membrane-bound IICBGlc (the glucose permease), and glu-cose. For other carbohydrates, specific enzymes II exist (with the A, B, and C domains pre-sent either as a single polypeptide or as multiple proteins, depending on the carbohy-drate that is transported), which accept the phosphoryl group from HPr. The below de-scription further elaborates the same.

In E.coli cells Glucose is transported through a transporter. PtsI (Enzyme I) is a cytoplas-mic protein that serves as the gateway for the phosphoenolpyruvate:sugar phos-photransferase system (PTSsugar) of E. coli K-12. There are two (PtsI and PtsH) sugar non-specific protein constituents of the PTSsugar. PtsI is one of them. PTS proteins, which along with a sugar-specific inner membrane permease protein, affect a phosphotransfer cascade. This cascade results in the coupled phosphorylation and transport of a variety of carbohydrate substrates.
PEP group translocation, also known as the phosphotransferase system or PTS, is a meth-od by which bacteria uptake sugar using the energy derived from phosphoenolpyruvate (PEP). This is a multicomponent system utilizing proteins from plasma membrane and some other proteins from cytosol. The PTS system uses active transport. Membrane transported proteins are modified by this mechanism.

The phosphotransferase system is involved in transporting many sugars into bacteria, in-cluding glucose, mannose, fructose and cellobiose. PTS sugars differ between bacterial groups, depending on the most suitable carbon sources available in the environment from where the bacteria had evolved. In E. coli import specificity has been determined by 21 different proteins. Of these, 7 to determine fructose (Fru) family, 7 for the glucose (Glc) family, and 7 belong to the other PTS permease families.
The phosphoryl group on PEP is eventually transferred to the imported sugar via several proteins. The phosphoryl group is transferred to the Enzyme E I (EI), Histidine Protein (HPr) and Enzyme E II (EII) to a conserved histidine residue. In case of Enzyme E II B (EIIB) the phosphoryl group is usually transferred to a cysteine residue, rarely to a histidine.
In the process of glucose PTS transport specific of enteric bacteria, phosphoryl group of PEP is transferred to a histidine residue on EI. EI, in turn, transfer the phosphate to HPr. From HPr the phosphoryl group is transferred to EIIA which is specific for glucose. EIIA further transfers the phosphoryl group to EIIB. Finally, EIIB phosphorylates glucose as it crosses the plasma membrane through the transmembrane Enzyme II C (EIIC), forming glucose-6-phosphate. While transferring the Glucose through the membrane, its phos-phorylated form Glucose 6 phosphate is beneficial because glucose is freely transferable through the membrane whereas its phosphorylated form is not. The HPr is common to the phosphotransferase systems of the other substrates mentioned earlier, as is the up-stream EI.
PtsI gene mutant, the deletion mutant, is not capable of synthesis of EI protein. EI is the entry of PEP phosphoryl group transfer cascade. Absence of this protein will result in loss of growth in glucose media (schematically represented in figure 1).
Deletion of this PtsI gene block the glucose metabolism machinery of E.coli cell. Host propagated in a minimal media like M9 media, having 0.4% glucose as a sole source of carbon, can’t survive if the Glucose metabolism pathway gets affected. Hence, deletion of the Pts1 gene makes the E.coli conditional lethal for the selective nutritional pressure. The same deletion strain was viable in Lysogeny broth(LB) media. Thus, the deletion con-struct wasobtained in LB media only. Selection of the same was done by replica plating in M9+ glucose media.
Pts1 gene was selectively deleted from the host genome by homologous recombination by Red /ET recombination system available from Gene Bridges. The central step in Red/ET recombination is the crossover step between a targeting construct containing homology arms (hm) and the target which can be a gene locus on the E. coli chromosome or any other stretch of DNA in a BAC or plasmid vector. Homologous recombination allows the exchange of genetic information between two DNA molecules. Gene Bridges Red/ET re-combination technology is based on homologous recombination involving a ? phage-derived protein pair, Reda/Redß, and 50 bp homology regions. Reda are 5‘- 3‘exonucleases, and Redß are DNA annealing proteins. The recombination is further as-sisted by lambda (?)-encoded Gam protein, which inhibits the RecBCD exonuclease activi-ty of E.coli.
Presence of the PtsI gene in the plasmid vector allowed us to screen the transformants of the PtsI deletion strains in M9+ glucose agar plate. Using this strategy as per Figure 2 we can substitute the conventional antibiotic based transformant screening method.
Since the sequence of the homology regions can be chosen freely, any position on a tar-get molecule can be specifically altered. The recombination process is strictly controlled due to an optimized design of the pRedET expression plasmid (schematically presented in figure 3). The genes for the recombination proteins are under the control of an inducible promoter and the plasmid carries a temperature sensitive origin of replication for easy curing of the plasmid after recombination.
Pts1 gene flanking primers were designed to replace chromosomal Pts1 gene with Kan/Neo cassette amplified from pKD13 vector (schematically presented in figure 4). 50bp long homology arms corresponding to the sequences flanking Pts1 gene on the chromo-some were added to the functional cassette by PCR. PCR with the following primers on pKD13 plasmid DNA amplified the Kan/Neo cassette flanked with Pts1 homology region. This linear product was used for homologous recombination using pRed/ET plasmid.
The gene of interest isolated by any conventional technique, such as PCR (Polymerase Chain Reaction), cloning or chemically synthesized.
· PCR reaction setup (in 50 µl) was as follows:
40 µl dH2O
5.0 µl 10 x PCR reaction buffer
1.0 µl 10 mM dNTP
1.0 µl Forward primer (10 µM)
1.0 µl Reverse primer (10 µM)
1.0 µl pKD13 plasmid (5ng)
1 µl SuperZym Taq DNA polymerase (2.5 U/µl)
• An annealing temperature of 57°- 62°C was optimal.
• PCR Profile: Initial denaturation step 30 sec 95°C; thirty cycles: 10 sec 95°C, 30 sec 55°C, 60 sec 72°C; final elongation step 10 min 72 °C.
• A 5 µl aliquot of the PCR product was electrophoresed on a 1% agarose gel to ensure the PCR was successful. The size of the PCR product for the Kan/Neo cassette is 1037 bp.
• The PCR product was column was purified.

The next step was transformation of pRed/ET plasmid in competent E. coli dH5alpha strain. The transformation protocol was as follows:
a) Competent cells were taken out of -80°C and thawed on ice (approximately 20-30 mins).
b) 2ng of pRed/ET plasmid DNA was mixed into 100 µL of competent cells in a mi-crocentrifuge tube by gentle tapping.
c) The competent cell/DNA was incubated mixture on ice for 20-30 mins.
d) Heat shock was given to each transformation tube placing in a 42°C water bath for 60 secs, followed by immediate chilling on ice for 10 min.
e) 700 µl of LB media (without antibiotic) was added to the bacteria and grown in 37°C shaking incubator for 45 min to 1hour.
f) The tubes were centrifuged at 300 rpm for 5 mins. The excess LB was decanted and pellet was resuspended in remaining LB and plated on LB agar plate con-taining Ampicillin.
g) Plates were incubated overnight at 30°C.

The colonies after transformation of host cell with pRed/ET plasmid on LA-Amp plate were as shown in figure 5.
Positive clones containing pRed/ET plasmid were made electro-competent to take up lin-ear PCR fragment by electroporation. The linear DNA fragment (Kan/Neo cassette) with homology arms that were used to replace the DNA fragment on the chromosome.
a) To start the procedure, overnight cultures was given in 3ml LB media contain-ing ampicillin from a single colony from the pRed/ET transformed plate. The cultures were incubated at 30°C overnight in a shaker incubator.
b) The next day, 10ml of LB broth was inoculated with 0.5% of primary culture and incubated at 30°C for approximately 3 h, shaking at 1100 rpm until OD600 ~ 0.4.
c) 10% L-arabinose was added to the secondary culture, giving a final concentra-tion of 0.3%-0.4%. This induced the expression of the Red/ET Recombination proteins. One part of secondary culture was left un-induced.
d) Both induced and un-induced secondary cultures were incubated at 37°C, shaking for 1 hour. At this step, it is important that cells are incubated at 37°C, the temperature at which all proteins necessary for the subsequent recombi-nation are expressed. Even though at 37°C curing of pRed/ET plasmid occurs, but any daughter cell still has on average 2-3 copies left and thus express the recombination proteins.
e) Preparation of the cells for electroporation:
· Before starting the procedure, 10% glycerol was chilled on ice for at least 2 hours. Benchtop centrifuge was cooled to 2°C and electro-poration cuvettes were also chilled.
· Both the secondary cultures were centrifuged for 30 sec at 11,000 rpm in a pre-cooled microfuge benchtop centrifuge (at 2°C). The supernatant was discarded by pellet was placed on ice and resuspended in 1 ml chilled 10% glycerol. The tube was centrifuged and the supernatant was discarded such that 50µl will be left in the tube with the pellet. Cells were resuspended and the tubes were kept on ice.
f) Further, 400ng of linear Kan/Neo cassette with homology arms was added to the pellet to each of the two microfuge tubes (induced and un-induced), and the mixture was pipetted into the chilled electroporation cuvettes.
g) Electroporation was performed at 1350 V, 10 ?F, 600 Ohms. 1 LB medium with-out antibiotics was added to the cuvette. The cells were mixed carefully by pi-petting up and down and pipette back into the microfuge tube. The cultures were incubated at 37°C with shaking for 3 hours. This process results in re-combination.
The culture was centrifuged for 30 seconds followed by removal of 900µl of thesupernatant and the cells were resuspended in the remaining medium. The cells were plated onto LB agar plates containing kanamycin (50 µg/ml).
The figure 6 shows induced and un-induced plates after electroporation with linear Kan/Neo cassette in pRed/ET transformed dH5 alpha cells.
The obtained colonies were further analyzed by colony PCR using Stellaris Colony PCR Master Mix for verification of successfully modified genome. The primers used for colony PCR are designed such that one primer located on the chromosome and the second one on the cassette, so a correctly recombined fragment yielded the expected PCR product confirming the correct insertion of the kanamycin/neomycin resistance cassette.
The procedure for colony PCR is as follows:
· PCR reaction setup: (for 20 µl)
8 µl dH2O
10.0 µl 2 x Stellaris Colony PCR Master Mix
0.5 µl Forward primer (10 µM)
0.5 µl Reverse primer (10 µM)
For template, single colony was directly picked up from the plate and added to the PCR reaction.
• PCR Profile: Initial denaturation step 15 mins at 95°C; thirty cycles: 10 sec 95°C, 30 sec 55°C, 30 sec 72°C; final elongation step 5 min 72°C.
· A 5 µl aliquot of the PCR product was electrophoresed on a 1% agarose gel to ensure the PCR was successful. The size of the PCR product for positive clone was 200bp repre-sented in the figure 7.
For confirmation of pts1 gene deletion; Pts1 gene in the E.coli K12 strain was replaced by Kanamycin resistant gene. Recombinant strains were initially selected on LB-Agar- Kana-mycin plate. Recombinant strains were screened for successful deletion of Pts1 gene. DNA was isolated from the Kan selected colonies. PCR using the Pts1 forward and reverse pri-mer were performed on each and individual genomic DNA. As a control of the PCR reac-tion and to ascertain the quality of the isolated genomic DNA, PCR reaction with 16S rDNA specific primers were also performed. Strains, giving amplification with 16S rDNA specific primers but not with Pts1 gene specific primers were selected as successfully Pts1 deleted strains. Another control PCR reaction was also performed. To confirm the quality of the Pts1 gene specific primers, BL21DE3 strains were propagated and genomic DNA was isolated from the culture. This genomic DNA was also used as a template for amplifica-tion with both the primers. Good amplification found on the genomic DNA was the one isolated from BL21DE3 cells (Figure 8).
Pts1 mutants were grown in M9 minimal media supplemented with 0.4% glucose as well as LB agar. Both the plates has Kanamycin selection. Since Pts1 deletion block the glucose metabolism machinery of E.coli, so cells propagated in a minimal media like M9 media, having 0.4% glucose as a sole source of carbon, can’t survive. Hence, deletion of the Pts1 gene makes the E.coli conditional lethal for the selective nutritional pressure. Host cell, transformed with a Kan resistant plasmid propagates in both M9 minimal media as well as LB media. When pts1 knockout strain was subjected with nutritional selection, the re-sults were as shown in figure 9.
Pts1 gene was replaced by Kanamycin gene. The wild type E.coli K12 is sensitive to Kana-mycin. The recombinants were selected in presence of Kanamycin. Deletion of the Pts1 gene was further confirmed by PCR with PTS1 gene specific primer. Absence of any ampli-fication in modified cell was further supported by the positive amplification in wild type cell and universal amplification with 16S rDNA specific primer. Hence, it was confirmed that the Pts1 gene has been successfully deleted from the E.coli K12 genome.

Vector Development
The vector was designed to express the Ranibizumab antibody. Its major components viz. pts1 gene, Ranibizumab heavy chain and light chain, LacI, Origin, AmpR gene (schemati-cally shown in Figure 10).
PtsI gene: PtsI gene (marked as EI in the Fig.10) involved in the phosphorylation of Glu-cose molecule entered in the cell. Deletion of the PtsI gene leads to the deficiency in phosphorylation of glucose metabolism and the deficient cells can’t grow on the M9 me-dia with Glucose as soul carbon source. The vector (SEQ ID No. 1) was designed in such a way that the PtsI gene was placed under a constitutive promoter. Promoter sequence used for expression of beta lactamase gene in the pUC vectorsystem was opted to ex-press the PtsI gene positioned in the new vector.
Ranibizumab heavy chain & light chain: Heavy chain and light chain of the Ranibizumab were designed as a polycistronic manner. Positioning the heavy chain and light chain in a same transcript helped to maintain the same stoichiometry of the peptides. Both pep-tides were expressed without any tag. To ensure the termination of the translation, two consecutive stop codons were positioned at the end of each coding DNA sequences. Pro-moter of the polycistronic RNA was used an inducible promoter for expression at a cer-tain growth phase. In this vector T7 Promoter/lac operator system was used. Termination of transcription was assured by a T7 terminator sequence.
LacI gene:LacI gene plays a crucial role in the control of gene expression in this vector. It controlled the expression of Ranibizumab. LacI encodes the repressor protein which blocks the lac operator sequence present downstream of the T7 promoter. After induc-tion with IPTG (Isopropyl ß-D-1-thiogalactopyranoside) binds with the LacI encoded re-pressor protein and release from the operator sequence. LacI sequence was selected from pET28b vector.
Origin:The replication origin of the vector kept as a high copy number origin. High copy ensures higher level of expression. To have a better expression profile, pBR322 replica-tional origin was used without the rop (repressor of primer) copy number control. The origin sequence was collected from pUC19 vector.
AmpR gene:Ampicillin resistant gene in the vector was for selection of the vector for ini-tial screening. When PtsI selection procedure worked, the AmpR gene was deleted from the construct.
Presence of the PtsI gene in the vector allowed us to screen the transformants of the PtsI deletion strains in M9+ glucose agar plate. Using this strategy as per Figure 2 we can sub-stitute the conventional antibiotic based transformant screening method.

Recombinant Protein Expression
E.coli BL21-DE3 strain was considered as a host cell for the recombinant protein expres-sion. A cascade pathway regulates Glucose transport to the E.coli cell and its metabolism. The Glucose enters the cell through the cell membrane. Unless and until Glucose gets phosphorylated, it remains permeable through the cell membrane. The phosphate group from Phospho Enol Pyruvate is transferred to glucose through a cascade pathway. The first enzyme involved in this pathway is a transferase, encoded by a gene named Pts1 (Phospho Transferase System, Enzyme 1). Deletion of this gene blocks the glucose metab-olism machinery of E.coli cell. Host propagated in a minimal media like M9 media, having 0.4% glucose as a sole source of carbon, can’t survive if the Glucose metabolism pathway gets affected. Hence, deletion of the Pts1 gene makes the E.coli conditional lethal for the selective nutritional pressure.
The same gene, under a constitutive promoter has been placed in the recombinant vec-tor DNA. The vector, after getting transformed in the E.coli expresses the PtsI gene which in turn complemented the deficiency of the host cell. Hence, only the transformants could grow in the minimal medium with Glucose as a soul nutrient source.

Cloning Details:
The vector DNA (SEQ ID No. 1) was constructed as a chimeric construct of a cloning vector and an expression vector. PtsI gene was kept under a constitutive promoter. The promter sequence was use as the same that exist in all commercially available ampR promoter. PtsI gene was PCR amplified from the E.coli BL21DE3 genomic DNA. The primers were de-signed with PstI and ClaI restriction sites at the upstream of forward and reverse primer respectively. Ranibizumab heavy chain and light chain polycistronic gene was synthesized by gene synthesis and finally amplified by two primers engineered with SacI and SalI at the upstream of forward and reverse primer respectively. The vector contained a LacIgene sequence which codedfor Lac repressor protein to inhibit the basal level expression from T7 promoter.
Beta-lactamase gene removal.
The Ampicillin resistant gene (beta-lactamase) from the vector was removed by inverse PCR method. Two inverse primers were designed flanking the Ampicillin resistant gene. PCR amplified product was treated with DpnI to remove trace amount of template DNA present. As the PCR amplified DNA does not have any methylation, it remains intact after digestion. DpnI digested DNA was purified from the reaction using UniPro Gel Ex/Cleanup Kit. 50ng of purified inverse PCR amplified DNA was phosphorylated using T4 Poly nucleotide kinase. End phosphorylated DNA was allowed to self ligate by T4 DNA Ligase. Self ligated DNA was transformed in BL21DE3?ptsIhost cell. Transformants were screened by selecting them on M9+Glucose media as shown in Figure 2. Re-plating the transformants on Luria agar plate added with Ampicillin, does not show any growth, confirming the successful removal of Ampicillin resistant gene.
Maintenance Host:
As no antibiotic resistant genes were present in the vector, no antibiotic resistance could be used for clone selection and maintenance. The nutritional selection pressure could be utilized by transforming the vector inBL21DE3?ptsIand propagating the transformants in M9+Glucose media.
Expression Host:
E.coli cells having T7 RNA Polymerase gene in the chromosome was compatible as expres-sion host for the designed vector. Expression has been checked in BL21DE3 cells. For ex-pression of cloned gene in antibiotic free selection pressure, the expression host was the developed host. The developed host cell has the PtsI gene deletion in BL21DE3 E.coli cell. The expression host should be recA1, endA1 & lon protease deficient.

Example 1
Production of Ranibizumab:
Ranibizumab is a monoclonal antibody fragment (Fab). It works as an anti-angiogenic. Ranibizumab has got its approval to treat the "wet" type of age-related form of vision loss commonly known as macular degeneration (AMD, also ARMD).
Heavy chain and light chain of the Ranibizumab were designed in a polycistronic manner. Positioning the heavy chain and light chain in a same transcript would help to maintain the same stoichiometry of the peptides. Both of the peptides are to be expressed with-out any tag. To ensure the termination of the translation, two consecutive stop codons are positioned at the end of each coding DNA sequences (CDs). Promoter of the polycistronic RNA has been used as an inducible promoter for expression at a certain growth phase. In this vector T7 Promoter/lac operator system was used. Termination of transcription has been assured by a T7 terminator sequence.
Construct Design:
The heavy chain and light chain of the antibody are designed to be synthesized in a polycistronic fashion to maintain the equal dose of expression of both the peptides. The polycistronic gene was placed under the T7 promoter with a tight control of LacI repres-sion. Each cistron are assured to be single peptide, rather not to be a continuous chin of amino acid, by two successive stop codons at the end of each reading frame.
The gene sequence to encode the amino acid sequence mentioned was codon optimized for best expression in E.coli maintaining the codon bias of the E.coli cells. A short peptide sequence was added at the N terminal region for assured delivery of the synthesized pro-tein at periplasmic space. Localisation of over expressed protein at periplasmic space helps to purify the protein without any non-specific endogenous protein.
Confirmation of mother construct by restriction digestion:
The main construct after development was digested with restriction enzymes. The major two genes cloned in the vector were screened. The final construct was digested with the respective enzymes to ascertain the desired drop down of cloned insert. PtsI gene clon-ing was confirmed by digestion with PtsI (type II restriction endonuclease) and ClaI (Cary-ophanon latum L site specific restriction endonuclease). After digestion in recommended buffer by manufacturer (Thermo) at 370C for 2h, a 1.7kb fragment was dropped down from the construct.
The clone confirmation of Ranibizumab polycistronic gene was done by restriction diges-tion with SacI and SalI. SacI restriction enzyme recognizes GAGCT^C sites whereas SalI re-striction enzyme recognizes G^TCGAC sites. In both the cases, 2mg of BIG_Final plasmid was digested with respective restriction enzymes in the manufacturer referred buffer sys-tem for 2hours at optimum temperature. After digestion, the enzymes were heat inacti-vated by incubating at 850C for 15 min. Reaction was electrophoresed on 1% agarose-TAE gel in presence of molecular weight marker.
Confirmation of recombinants by Protein expression
The recombined vector was transformed in the modified BL21 DE3 host. Single colony from the overnight grown plate was inoculated in 3ml LB medium and allowed to grow up to saturate level. Secondary inoculums given in 5ml of LB broth with volumetrically 0.1% of the secondary culture volume by the initially grown cells. Inductions were checked in four aliquots, 0.4mM, 0.5mM, 0.75M and 1.0mM at 370C for 2h at 140rpm speed of shaking. A clear induction observed in the gel almost all the induction IPTG con-centrations.
The ladder did not run in the penultimate lane, hence the position of marker is shown as numerical value of the molecular weight of the corresponding band of ladder.

The present invention provides a platform cloning technology which leads to a scalable fermentation process and higher yield. The regulatory hurdles will be low due to antibi-otic free production process starting from clone till final product.

,CLAIMS:We Claims,
1. A process of selective nutritional pressure for producing a recombinant protein, wherein the process comprises of following steps:
a) knocking out of gene from host cell by process of homologous recombination,
b) transforming host cell with a vector comprising a gene encoding the recombi-nant protein and knocked-out gene placed under a constitutive promoter,
c) growing the transformed host cells of step b in minimal media,
d) obtaining recombinant protein expressed.
2. The process of claim 1, wherein the host cell is prokaryotic cell.
3. The process of claim 3, wherein the host cell is Escherichia coli.
4. The process of preceding claims, wherein the E. coli strain is K-12.
5. The process of claim 1, wherein the vector has sequence ID No. 1.
6. The process of preceding claims, wherein the transformed host cell is deficient in glucose metabolism in post transportation phosphorylation cascade pathway.
7. The process of preceding claims, wherein the knock-out gene in host cell is pts1.
8. The process of preceding claims, wherein the knock-out gene in host cell is selec-tively deleted by Red /ET homologous recombination system.
9. The process of preceding claims, wherein Pts1 gene is replaced by Kanamycin re-sistant gene.
10. The process of preceding claims, wherein Pts1 gene flanking primers are designed to replace chromosomal Pts1 gene with Kan/Neo cassette amplified from pKD13 vector.
11. The process of claim 1, wherein, the media used for growth of transformed host cell is M9 with glucose as sole carbon source.
12. The process of preceding claims, wherein recombinant protein expressed is Gcsf, interferon, parathyroid hormone, interleukin, reteplase, thrombolytic agent, anti-body, insulin and its analogues.
13. The process of preceding claims, wherein recombinant protein expressed is a monoclonal antibody.
14. The process of preceding claims, wherein recombinant protein expressed is Ranibizumab.
Dated this 26th day of July, 2018

Signature: ______________________
Name: Mohsin R. Arabiani
(Agent of the applicant)