CELL THEREOF"

TECHNICAL FIELD

The present disclosure relates to the field of Biotechnology. Particularly, it relates to recombinant DNA technology, wherein vector and enzyme free cloning (VEF-C) method is presented which results in custom plasmids harbouring desired fragments of interest in recA- null strains of host cells, such as E.coli. Moreover, the present invention is an economical and time effective alternative as against conventionally used cloning methods as well as existing ligase-independent cloning systems.

BACKGROUND

Genetic recombination occurs in all forms of life and involves exchange of nucleic acid sequences between two chromosomes or DNA molecules. In prokaryotes, RecA is the key enzyme in performing homologous recombination, whereas heteroduplexes are formed when one strand from each parental duplex DNA is exchanged. However, in cloning strains recA activity is disadvantageous as homologous regions between plasmids can recombine and form undesired products. Therefore, a recAl mutation is generally performed in bacterial strains (example - DH5a, JM109) to reduce recombination by 10,000 fold known in the prior art. However, as recAl can still bind to single stranded DNA and form complementary strand renaturation, a more stringent system for cloning DNA is required. For many years, ligase independent cloning (LIC) has been carried out in recAl strains; however the monomeric maintenance of plasmids is 1 : 10,000. Formation of dimers would drastically reduce copy number; produce intermediate undesired products, etc. The existing pathways in E.coli are described below for further understanding.

In general, the recBCD pathway is the preferred pathway for homologous recombination in E.coli. Here, three subunits namely recB, recC and recD form a heterotrimeric complex that acts as a helicase/nuclease unwinding and degrading duplex DNA. The recB subunit possesses 3 '-5' helicase activity, recD acts as a 5 '-3' helicase and the nuclease function is sequestered in the recB domain. Upon encountering a recombination hotspot (Chi), the recBCD enzyme (~ 10 in numbers) stops degrading both 5' and 3' strand and switches its motor to create a 3' extension. This single stranded DNA is protected by SSB proteins (which is loaded by recBCD), which inhibits homologous recombination by recA (competitively) and avoids the action of helicases and nucleases on the ssDNA. Interestingly, recA is also loaded onto the strand by recBCD itself thus displacing SSB, making it a key player in assisting the recA biochemistry in-vivo. The recA protein replaces SSB from the single strand in the form of a polymer, scans for homology and performs the strand exchange reactions. Thus, recA involvement is vital functioning of the recBCD pathway.

Unlike the recBCD enzyme, which functions as a heterotrimeric complex, the recFOR pathway incorporates the function of recF, recO and recR separately. The recFOR pathway plays a secondary role, occurring in the absence of recBCD. Here, recQ helicase and recJ exonuclease are two enzymes involved in generating ssDNA from dsDNA to facilitate recombination. The RecO protein binds to both dsDNA and ssDNA and promotes recA- mediated renaturation of complementary strands of DNA. This reaction is enhanced when recO forms complexes with SSB. Further, recO combines with recR to make a recOR complex that loads recA onto the SSB. Here, The recF protein binds to dsDNA in the presence of ATP in a stoichiometry of 1 recF per 15 nucleotides and has been proposed to direct recA to the single strand gaps in dsDNA. RecO and recRact as mediator proteins that help overcome to SSB inhibition in the bacterial cell. Further, recA displaces the SSB and performs homology search and finally completes strand exchange. Till date, the prior art suggests that recF interferes with recOR function and strongly competes with recO for interacting with recR. This indicates that recF may work with recOR complex in some processes and individually in others. Thus, explaining the inconsistency of recF, recO and recR in the genomes of different bacteria. Furthermore, the overexpression of recOR suppresses effects of recF overexpression, which means they have to be regulated systematically at each step in recombination for them to function together. If any of the above mentioned enzymes are missing or un-regulated, it could lead to the elimination of the pathway itself. RecA being a key player in the above pathway, its deletion leads to a recFOR and recBCD null phenotype.

The recE pathway is specifically only activated in recB and recC mutants by additional mutations in sbcA. These mutations lead to the activation of the recE gene, which encodes for a 96-kDa 5'-3' double strand exonuclease (Exo VIII) and recT which is a single strand DNA binding protein. Although conjugational recombination in the recE pathway is recA dependent, recombination of plasmids requires only recE and not recAl but, in a strain that lacks the sbcA (repressor of recE) mutation, this pathway is ruled out. After generating single strands by recE, in a mg2+ dependent and ATP-independent manner the annealing of complementary strands is facilitated by recTl . However, no clear evidence on the ssDNA generation in case of closed plasmids has been elucidated, as there is no entry point for a single strand exonuclease like recE termed as ET recombination, a minimum of 60-100bp was proposed to be showing homologous recombination which is increased with increase in the base pairs involved in annealing. To add to its complexity, it was shown that mutations in recO, recR and recJ drastically reduced recE-mediated recombination. This suggests that recFOR and recE pathways may overlap; however in the presence of recBCD and absence of recA, both remain inactive.

In the absence of necessary enzymes (recA), bacteria tend to display an adaptive characteristic when subjected to survival threatening situations. Such situations are similar to SOS response that results in the expression of various genes into the cytoplasm when triggered by external stress.

The present disclosure simulates such a situation in-vivo and exploits it to facilitate seamless recombination of fragments that results in custom plasmids in a recA null strain. ET recombination and ligase independent recombination require pre-treatment with greater than 60 and greater than 24bp overhangs, respectively. The present invention uses as less as 15bp or more of homology to get monomeric plasmids containing the gene of interest/fragments without any pre-treatment of DNA fragments (Figure 1). Due to its flexibility, the instant disclosure demonstrates the use of this technique in generating customized plasmids of any gene or fragment desired. SUMMARY OF THE DISCLOSURE

The present disclosure involves an enzyme and vector free cloning method/strategy that results in custom plasmids harbouring desired gene of interest (GOI), wherein the DNA fragments are transformed directly into host systems without sub-cloning or restriction digestion. This facilitates cloning to expression in two days as compared to conventionally used strategies which are prolonged and error prone.

The present disclosure relates to a method of obtaining vector from linear DNA fragment, said method comprising acts of - a) generating one or more linear DNA fragment with overhangs and optionally treating with Dpnl and amplifying the DNA fragment, b) transforming the said fragment into host cell to obtain transformed cell, and c) incubating said transformed cell for joining of the one or more DNA fragment and obtaining the vector.

The present disclosure also relates to a vector comprising one or more linear DNA fragment having overhangs, origin of replication and antibiotic marker, wherein the DNA fragments join to produce the vector.

The present disclosure relates to a method of obtaining transformed cell from linear DNA fragment, said method comprising acts of - a) transforming a host cell with the one or more linear DNA fragment having overhangs and b) incubating said cell for joining of the one or more DNA fragment, followed by antibiotic screening to obtain the transformed cell.

The present disclosure also relates to a transformed cell comprising one or more linear DNA fragment having overhangs, wherein fragments join to produce a vector within the cell.

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:

Figure 1 depicts schematic representation of Vector and enzyme free Cloning (VEF-C).

Figure 2 depicts customisation of plasmid with gene of interest (VEF-C).

Figure 3 depicts vector map for luciferase (~700bp) cloned into petl5b (5.3kb) in accordance with the present invention for 2 fragment joining.

Figure 4 depicts vector map for fragment A (~300bp) and vector backbone (~2.1Kb) in accordance with the present invention for 2 fragment joining.

Figure 5 depicts vector map for fragment A (~300bp), B(2Kb) and vector backbone (~2.1Kb) in accordance with the present invention for 3 fragment joining.

Figure 6 depicts vector Map of CKEM (product of ColEl, Kanamycin, eGFP, MBP) in accordance with the present invention for 4 fragment joining.

Figure 7 depicts confirmation of SIM (template free). Figure 8 depicts confirmation of SIM (Electron-microscopy). DETAILED DESCRIPTION

The present disclosure relates to a method of obtaining vector from linear DNA fragment, said method comprising acts of:

a. generating one or more linear DNA fragment with overhangs and optionally treating with Dpnl and amplifying the DNA fragment;

b. transforming the said fragment into host cell to obtain transformed cell;

c. incubating said transformed cell for joining of the one or more DNA fragment and obtaining the vector.

The present disclosure also relates to a vector comprising one or more linear DNA fragment having overhangs, origin of replication and antibiotic marker, wherein the DNA fragments join to produce the vector.

In an embodiment of the present disclosure, the vector is produced by method as above.

In another embodiment of the present disclosure, the overhangs of the one or more DNA fragment comprise homologous sequence of atleast 15bp length.

In yet another embodiment of the present disclosure, the DNA fragment is generated and amplified by subjecting to PCR amplification; wherein the treating with Dpnl is carried out at temperature of about 37°C, for time duration ranging from about 1 hour to about 4 hours; and wherein the Dpnl treatment is followed by gel extraction using about 0.1 to about 3% Agarose; and PCR amplification.

In yet another embodiment of the present disclosure, the transformation is carried out by technique selected from group comprising heat-shock and electroporation or combination thereof.

In yet another embodiment of the present disclosure, the DNA fragment is linear and template-free with size ranging from about 100 bp to about 6000 Kb, and is selected from group comprising marker, antibiotic marker, origin of replication, fusion tag, affinity tag, gene of interest, promoter region, gene encoding signal sequence and regulatory region or any combinations thereof.

In yet another embodiment of the present disclosure, the host cell is recA-defective host cell. In yet another embodiment of the present disclosure, the host cell is E.coli.

In yet another embodiment of the present disclosure, the one or more DNA fragment join to result in circular and monomeric plasmid. In yet another embodiment of the present disclosure, the method is applied to 2, 3 or 4 DNA fragments or combinations thereof, to result as the vector by applying external stress using bacterial antibiotic selected from group comprising kanamycin, ampicillin and chloramphenicol or any combinations thereof.

In yet another embodiment of the present disclosure, the transformed cell is incubated at temperature of about 37°C for time duration ranging from about 1 hour to about 1.5 hours. In yet another embodiment of the present disclosure, the method is an enzyme and vector free method.

In yet another embodiment of the present disclosure, the DNA fragment to be generated includes selection marker or antibiotic marker and origin of replication.

The present disclosure relates to a method of obtaining transformed cell from linear DNA fragment, said method comprising acts of:

a. transforming a host cell with the one or more linear DNA fragment having overhangs; and

b. incubating said cell for joining of the one or more DNA fragment, followed by antibiotic screening to obtain the transformed cell.

The present disclosure also relates to a transformed cell comprising one or more linear DNA fragment having overhangs, wherein fragments join to produce a vector within the cell.

In an embodiment of the present disclosure, the cell is produced by method as above.

In another embodiment of the present disclosure, the transformation is carried out by technique selected from group comprising heat-shock and electroporation or combination thereof.

In yet another embodiment of the present disclosure, the transformed cell is incubated at temperature of about 37°C for time duration ranging from about 1 hour to about 1.5 hours. In yet another embodiment of the present disclosure, the method is an enzyme and vector free method.

The present disclosure involves an enzyme and vector free cloning strategy/method that results in vector/plasmids harbouring desired gene of interest (GOI). The instant method gives flexibility to the user to employ desirable DNA fragments such as markers, promoters, kind of antibiotic marker, origins of replication, fusion tags, affinity tags, gene encoding signal sequence, regulatory region etc along with desired GOI, which is transformed directly into expression host systems without sub-cloning or restriction digestion (Figure 2). This facilitates cloning to expression in two days as compared to conventionally used strategies which are prolonged and error prone. Moreover, optimising protein expression is easier in such a strategy as different fragments are pooled to obtain various combinations. Upon expression of colonies, the highest expressing clone is identified and plasmid is isolated.

Another embodiment of the present disclosure is that inter and intra plasmid recombination is completely abolished, therefore no negative colonies are observed.

Yet another embodiment of the instant disclosure is the generation of vectors for various applications including other host systems like Yeast, Mammalian systems, cell-free expression systems etc.

In an embodiment, strains that do not contain any mutations of endo or exo nucleases, but only a recA-null mutation, all pathways would be inactive as described in the prior art. Without the homology search of recA, finding and annealing homology is impossible. In 1965, Clark et al., created a recombination deficient mutant of E.coli BL21. This mutant was incapable of combining DNA into its genome and was highly sensitive to UV radiation. This finding is still used in molecular cloning for preventing multimer formation and homology recombination between two/more identical plasmids or DNA fragments in-vivo of the same. This finding, led to the discovery of the existence of recA-independent pathways in E.coli. Upon closely observing the structure of recA and comparing it with recAl, the single strand binding ability of recAl still exists. Although it cannot bind to dsDNA like its wild type counterpart, it has been shown that complementary strand renaturation by Mg2+ mediated pathway is still possible in a recAl environment, thus explaining the 10,000-fold chance of obtaining recombination. This indicates that recombination is not completely abolished, but merely inhibited.

The present disclosure not only overcomes this 10,000-fold recombination event but also facilitates generation of customized plasmids based on user's choice without using any vectors or enzymes. As the cloning is also performed in an expression strain, shuttling of vectors or sub cloning is not required and protein expression is carried out, directly. This is time and cost effective as users can design/create a plasmid that meets their requirements.

The present disclosure involves a vector and enzyme free cloning (VEF-C) method/strategy that results in custom plasmids harbouring desired fragments of interest in recA-null strains of E.coli. This includes antibiotic resistant markers, promoters, origin of replication, fusion tags and thereof. The gene of interest is transformed directly into expression host systems without sub-cloning or restriction digestion, the strategy is time and cost effective (Figure 2).

In an embodiment, by employing external stress like bactericidal antibiotics, a survival- threatening situation is simulated in bacteria by creating a stress induced mechanism (SIM), which in turn adaptively expresses necessary proteins in its cytoplasm for facilitating 2, 3 and 4 fragment recombination. These proteins are exploited for enzyme free cloning of different fragments to result in circular monomelic and stably maintained plasmids/vectors harbouring the gene of interest.

In an embodiment, the joining of two fragments containing homologous overhangs is performed in recA-null strain. A gene of interest is PCR amplified using primers that 15-21bp overlaps with the plasmid. After amplification, both fragments are Dpnl treated and gel extracted. This product is further PCR amplified, if required and transformed into a recA-null strain of E.coli. Conventional protocol is followed for carrying out transformation, viz. heat- shock and electroporation.

In another embodiment, similar protocol is followed for the joining of four fragments and equimolar DNA concentration is maintained in all cases. This is different from existing techniques like ET recombination; which uses a minimum of 60-100bp homology to result positive clones and required recE and recT proteins for its functioning. Ligase independent techniques use T4 DNA polymerase to generate overhangs and subsequent cloning. However, the present disclosure does not employ any enzyme or vectors to generate fully functional plasmids in-vivo using 15-24bp homology. The strategy employed in Figure 1 is that PCR amplification is used to provide overhangs which are further followed by clean up. Next, these fragments are directly transformed into E.coli.

In another embodiment, as the present invention is performed in expression strain directly, the instant method facilitates 'cloning to expression' in two days as compared to conventionally used strategies, which are prolonged and error prone. Moreover, optimizing protein expression is easier in such a method as different fragments can be pooled to obtain various combinations. Upon expression of clones, the highest expressing clone is identified and plasmid isolated. Inter and intra plasmid recombination is completely abolished therefore no negative colonies are observed.

In another embodiment, the plasmids/vectors generated in the instant method has application in the generation of vectors for other host systems like Other E.coli strains, Yeast, insect cells, mammalian systems, cell-free expression systems etc.

In another embodiment, the instant methodology broadly employs the following acts:

1. Generation and amplification of DNA fragment (s) by polymerase chain reation (PCR).

2. Gel Extraction of fragments appearing at expected size/Dpnl treatment.

3. Generating larger quantity of PCR products.

4. Purification of PCR products.

5. Transformation into host cells for in-vivo cloning.

6. Screening of clones by colony PCR/restriction digestion and sequencing.

7. End applications like protein expression.

In an embodiment, the one or more DNA fragment with overhangs is generated in the instant method by PCR Amplification.

In another embodiment, the nucleic acid molecules or DNA fragments are obtained by amplification and are isolated with gel extraction by Agarose Gel Permeation method using about 0.1 to about 3% Agarose at about 20 V to about 100V by electrophoresis.

In yet another embodiment of the present disclosure, the stability of vector/plasmids obtained by the instant method are observed to be consistent for more than one generation. In yet another embodiment, the instant method is followed to obtain vector/plasmid which has application in the generation of vectors for other host systems like E.coli strains, Yeast, insect cells, mammalian systems, cell-free expression systems etc.

In another embodiment, in the instant method, the mechanism of recombination followed is other than that observed for recBCD, recFOR and recE pathways mediated by recA.

The invention involves a mechanism of recombination other than the known pathways in E.Coli (recBCD, recFOR and recE). Still further, the present invention is an economical and time effective alternative as against conventionally used cloning methods as well as existing LIC based systems.

The present disclosure is further elaborated with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.

EXAMPLES

Example 1 - Designing of Construct and primers:

'Primers for insert' for overhang generation: Overhangs is generated by designing primers such that desired fragments are arranged as shown in figure. 2. Below illustration shows the design of the forward primer with a fragment's nucleotides (marked as X, 21-24 bp) and sequence of the flanking region (marked as N, 21-24 bp):

5 'XXXXXXXXXXXXXXXXXXXXXNNNNNNNNNNNNNNNNNNNNNNNNNNN-3'

To design the reverse primer, the same strategy is applied, however nucleotides from the desired gene is reverse complemented, followed by flanking regions homologous to the succeeding sequence. Below illustration describes the design of the reverse primer:

3 ' -NNNNNNNNNNNNNNNNNNNNNNNNNNXXXXXXXXXXXXXXXXXXXXX-5 '

Example 2 - PCR Amplification of fragments/Generation of DNA fragments

PCR reactions are performed in gradient to identify appropriate annealing temperatures. A final concentration of IX PCR buffer, 200uM dNTPs, 0.5 μΜ of forward and reverse primer each, 1-5% DMSO, 5-10 ng of template DNA, 0.25-1 unit of DNA polymerase are used and made upto 25 μΐ with ultra-pure water. Any of these reagents are subjected to optimization, in case of non-amplified fragments. Company specific protocols are followed for generating fragments by PCR.

For obtaining pure PCR product, PCR clean-up is performed, which is clean up of target linear nucleic acid products and removal of non-specific products. If required, Dpnl treatment is performed by adding 1 unit in 25 μL PCR mix and samples are confirmed on a 0.2- 2% agarose gel. The Dpnl treatment is carried out at temperature of about 37°C, for time duration ranging from about 1 hour to about 4 hours. Example 3 - Transformation and Electroporation

For efficient transformation the following parameters are taken into consideration. All fragments are added in equimolar concentration (20-100ng each) to 30-40μ1 of chemically competent cells. After 30 minutes incubation, cells are heat shocked for 45 seconds at 42°C. Further, 1ml SOC is added after 2 minute incubation and allowed to grow at 37°C for 1-2 hour.

Electroporation is employed with the same concentration of reagents, wherein voltage is optimized across a range of 1500-5000V, whereas capacitance is kept constant. After incubating for 1-2 hour with SOC at 37°C, the cells are plated on LB agar containing respective antibiotic resistance (Kanamycin 25-30 μg/mL, ampicillin 40-50μg/mL and chloramphenicol 10-15μg/mL) and incubated at 37°C overnight (about 12-16 hours). Resulting colonies are picked, grown in LB with appropriate antibiotics and mini-prepped. Example 3.1

As depicted in Figure 1, joining of two DNA fragments containing homologous overhangs is performed in recA-null E.coli strain. A gene of interest - Luciferase is PCR amplified using primers that 15-21bp overlaps with the plasmid pET15b. After amplification, both fragments are Dpnl treated and gel extracted. This product is further PCR amplified, if required, and transformed into a recA-null strain of E.coli.

Conventional protocol is followed for carrying out transformation, viz. heat-shock and electroporation. The plates are incubated for about 12 hours to about 16 hours or overnight and colonies are picked, grown in LB containing appropriate antibiotic and plasmid isolated to obtain desired plasmid harbouring gene of interest.

Luciferase is cloned into pET15b such that they are not generated by conventional ligation methodology or any other means thereof. The insert is cloned such that no other form of strategy other than the present invention can be employed to generate such a fragment as depicted in Figure 3.

Example 3.2

Figure 4 is an illustration for a plasmid containing -300 bp gene of interest and ~2.1Kb vector backbone bearing ampicillin resistant marker is generated to demonstrate 2 fragments joining by employing present invention. The combination and overhanging regions used to make the VEFC possible are novel.

Example 3.3

Figure 5 is an illustration for a plasmid containing fragment A (-300 bp), fragment B (-2Kb) and ~2.1Kb vector backbone bearing ampicillin resistant marker is generated to demonstrate 3 fragments joining by employing present invention. The combination and overhanging regions used to make the VEFC possible are novel. Example 3.4

As depicted in figure 6, a plasmid containing ColEl (origin of replication), eGFP (gene of interest), Maltose binding protein (fusion tag) and Kanamycin resistant marker is generated by the present invention. The combination and overhanging regions used to make the VEFC possible are non-existent but not sequence specific.

Example 4 - Confirmation of stress induced mechanism (SIM)

Two identical 4 fragment recombination(s) (4FR) are performed. One is treated with Dpnl to remove template plasmid and the other is left as is i.e. bearing circular plasmid conferring similar resistance. Here, sample bearing circular plasmid + 4FR fragments did not result in SFM (Figure 7). However, without template, SFM is successful thus resulting in 4FR plasmids. This confirms the need for stress to facilitate recombination of 4FR fragments (Figure 2). SIM is further confirmed by electron microscopy, where cell wall thickening is observed (Figure 8).