GENETIC ENGINEERING WITH DEINOCOCCUS RADIODURANS FIELD The present disclosure relates in general to genetic engineering with Deinococcus radiodurans (D. radiodurans). BACKGROUND D. radiodurans is a polyextremophile bacterium that is well-known for its resistance to damage caused by ionizing and UV radiation [1,2], desiccation [3], and the vacuum of space [4– 6]. Its survivability has been the focus of many studies and has primarily been attributed to efficient protection against proteome degradation, ensuring cellular recovery and repair of damaged DNA [7]. The ability to function under extreme environments and stress makes D. radiodurans an attractive candidate chassis for biotechnology and industrial applications as it can withstand the physiochemical stress of industrial processes. Currently, this organism is used for bioremediation [8,9], small molecule production [10], and pigment production [11]. The D. radiodurans R1 genome was first sequenced in 1999 [12] and revealed that it is composed two chromosomes (DR_Main [2.65 Mbp] and DR412 [412 kbp]), one megaplasmid (MP1; DR177 [177 kbp]), and one small plasmid (46 kbp; CP1), that have 67% G+C content [12]. It has since been resequenced using PacBio sequencing and comparisons to the original published sequence identified large insertions and single nucleotide polymorphisms (SNPs) [13]. Available genetic tools include several shuttle plasmids [14–16], selection markers [17– 19], characterized promoters for inducible and tunable gene expression [20,21], and methods for generating gene deletions including a Cre-lox system [22]. Exogenous DNA can be introduced into D. radiodurans by natural transformation, chemical transformation and electroporation [23]. However, all these DNA delivery methods have low transformation efficiencies, possibly due to the presence of active restriction modification systems. The transfer of large plasmids or whole chromosomes has not yet been demonstrated in D. radiodurans. D. radiodurans is also polyploid containing up to 10 copies of its genome [24], making homogenous genetic modification more difficult [25]. Despite being one of the first bacteria to be sequenced and its remarkable ability to repair and reassemble genomic DNA in vivo, whole-genome engineering tools still do not exist for D. radiodurans. Synthetic cells based on mycoplasma [26,27] and E. coli [28,29] were created, but such accomplishments still have to be achieved for D. radiodurans. Conjugative plasmids are an attractive tool to alter or modify bacteria because conjugative plasmids have broad host ranges, are generally thought to be resistant to restriction-modification systems, are easy to engineer with large coding capacities, and do not require a cellular receptor that would provide a facile mechanism for bacterial resistance. Conjugation is also a useful tool for streamlining the genetic manipulation of organisms as it eliminates the need for high yield DNA extraction and has proven to be a versatile and simple method of DNA transfer capable of moving large plasmids to bacteria and eukaryotic organisms [30–36]. SUMMARY In one embodiment, the present disclosure provides for a genetically modified Deinococcus radiodurans (D. radiodurans), wherein the genetically modified D. radiodurans comprises at least three restriction enzyme genes knocked out, or at least four restriction enzyme genes knocked out, or at least five restriction enzyme gene knocked out, or all restriction enzyme genes knocked out. In one embodiment of the genetically modified D. radiodurans, the restriction enzymes gene include Mrr, Mrr2, ORF2230, ORF14075, ORF15360 and McrBC. In another embodiment of the genetically modified D. radiodurans, the genetically modified D. radiodurans lacks at least one of: MP1 megaplasmid, CP1 small plasmid, Chr 1 chromosome and Chr 2 chromosome. In another embodiment of the genetically modified D. radiodurans, the genetically modified D. radiodurans is derived from parental bacterium D. radiodurans R1. In another embodiment of the genetically modified D. radiodurans, the genetically modified D. radiodurans is free of exogenous or foreign nucleotide segments in the genome of D. radiodurans. In another embodiment, the present disclosure relates to a method of seamless deletion of a target nucleotide fragment of Deinococcus radiodurans (D. radiodurans), the method comprising: (a) transferring a DNA molecule to D. radiodurans, the DNA molecule having a seamless deletion (SD) cassette including in a 5’ to 3’ order (A) a first homology region to a first region in the genome of D. radiodurans, (B) a restriction endonuclease site that is not present in D. radiodurans (unique restriction endonuclease site), (C) a gene for a first selectable marker, (D) another homology region to the first region in the genome of D. radiodurans, the another homology region having less nucleotides than the first homology region, and (E) a second homology region to a second region in the genome of D. radiodurans, wherein the target nucleotide fragment is between the first region in the genome of D. radiodurans and the second region in the genome of D. radiodurans; (b) selecting D. radiodurans for the selectable marker, thereby obtaining D. radiodurans that include the SD cassette and deletion of the target nucleotide fragment; and (c) transferring a replicating plasmid to the D. radiodurans, the replicating plasmid including (i) a second selectable marker that is different from the first selectable marker and (ii) a gene encoding a restriction endonuclease that recognizes the unique restriction endonuclease site, whereby expression of the restriction endonuclease in the selected D. radiodurans cuts the unique restriction endonuclease site to obtain transconjugants of D. radiodurans with seamless deletion of the target nucleotide fragment. In one embodiment of the method of seamless deletion, the DNA molecule is a transmissible, non-replicating plasmid, and wherein step (a) comprises transferring the transmissible, non-replicating plasmid by conjugation from a donor cell to D. radiodurans. In another embodiment of the method of seamless deletion, the transmissible, non- replicating plasmid is a transmissible, non-replicating cis-conjugative plasmid having genes for conjugation. In another embodiment of the method of seamless deletion, the transmissible, non- replicating plasmid is a transmissible, non-replicating plasmid including an origin of transfer (oriT), and wherein the donor cell further includes a non-transmissible plasmid that includes genes necessary for conjugation in trans of the transmissible, non-replicating plasmid. In another embodiment of the method of seamless deletion, the donor cell is E. coli or Sinorhizobium meliloti. In another embodiment of the method of seamless deletion, the DNA molecule is a linear DNA molecule. In another embodiment of the method of seamless deletion, the SD cassette further includes a gene of interest between the another homology region and the second homology region, and wherein the transconjugants of D. radiodurans with seamless deletion of the target nucleotide fragment includes the gene of interest. In another embodiment of the method of seamless deletion, the gene for the first selectable marker and the gene for the second selectable marker confer D. radiodurans resistance to the first selectable marker and to the second selectable marker. In another embodiment of the method of seamless deletion, the DNA molecule further comprises a visual marker or a supplemented (auxotrophic) marker. In another embodiment of the method of seamless deletion, the gene for the first selectable marker comprises: (i) an antibiotic resistance gene for selection, and (ii) a gene for a screening marker that confers an optical signal to the D. radiodurans having the SD cassette. In another embodiment of the method of seamless deletion, the target nucleotide fragment is a gene, a restriction endonuclease site, a promoter, or an intragenic region. In another embodiment of the method of seamless deletion, the target nucleotide fragment is at least one restriction enzyme gene. In another embodiment, the present disclosure provides for a cell extract of the genetically modified D. radiodurans of the present disclosure. In another embodiment, the present disclosure provides for a use of the genetically modified D. radiodurans of the present disclosure in the treatment of cancer, anti-aging, production of antioxidants, production of carotenoid, production of fragrances, production of biofuels, bioremediation, small molecule production or pigment production. In another embodiment, the present disclosure provides for a method for delivering a DNA fragment of interest to Deinococcus radiodurans (D. radiodurans), comprising: (a) transferring by conjugation a transmissible plasmid from a donor cell to D. radiodurans, the transmissible plasmid including the DNA fragment of interest and a selectable marker; and (b) selecting D. radiodurans for the selectable marker, wherein the selected D. radiodurans include the DNA fragment of interest. In one embodiment of the method for delivering a DNA fragment of interest to D. radiodurans, the transmissible plasmid is a transmissible cis-conjugative plasmid having genes for conjugation, a gene for a selectable marker, and the DNA fragment of interest. In another embodiment of the method for delivering a DNA fragment of interest to D. radiodurans, the transmissible plasmid including an origin of transfer (oriT), the gene for the selectable marker, and the DNA fragment of interest, and wherein the donor cell includes a non- transmissible plasmid that includes genes necessary for conjugation in trans of the transmissible plasmid. In another embodiment of the method for delivering a DNA fragment of interest to D. radiodurans, the transmissible plasmid further includes an origin of replication for D. radiodurans. In another embodiment of the method for delivering a DNA fragment of interest to D. radiodurans, the transmissible plasmid comprises SEQ ID NO: 1. In another embodiment of the method for delivering a DNA fragment of interest to D. radiodurans, the non-transmissible plasmid is pTA-Mob or any other helper conjugative plasmid. In another embodiment of the method for delivering a DNA fragment of interest to D. radiodurans, the donor cell is a microbe or unicellular organism. In another embodiment of the method for delivering a DNA fragment of interest to D. radiodurans, the donor cell is E. coli or Sinorhizobium meliloti. In another embodiment of the method for delivering a DNA fragment of interest to D. radiodurans, the gene for the selectable marker confers D. radiodurans resistance to the selectable marker. In another embodiment of the method for delivering a DNA fragment of interest to D. radiodurans, the gene for the first selectable marker comprises: (i) an antibiotic resistance gene for selection, and (ii) a gene for a screening marker or a supplemented (auxotrophic) marker for visual screening. In another embodiment of the method for delivering a DNA fragment of interest to D. radiodurans, the DNA fragment of interest is a gene, a restriction endonuclease site, a promoter, or an intragenic region. In another embodiment of the method for delivering a DNA fragment of interest to D. radiodurans, the donor cell is E. coli ECGE101 DdapA, and wherein step (a) comprises (i) mixing the donor cell with the D. radiodurans and spreading the mixture on a conjugation plate having tryptone glucose yeast (TGY) media supplemented with diaminopimelic acid; (ii) incubating the conjugation plate; and (iii) scraping the cells off with TGY media to obtain a cell suspension; and wherein step (b) comprises plating the cell suspension in media supplemented with the selectable maker to select D. radiodurans transconjugants that include the DNA fragment of interest. In another embodiment of the method for delivering a DNA fragment of interest to D. radiodurans, step (b) further comprises plating the cell suspension in nonselective media without the selectable marker to determine the conjugation frequency. In another embodiment, the present disclosure provides for a method of cloning the whole megaplasmid (MP1) or whole small plasmid (CP1) of Deinococcus radiodurans (D. radiodurans) comprising: (a) transferring by conjugation a transmissible nonreplicating plasmid from a donor cell to D. radiodurans, the transmissible plasmid including an origin of transfer (oriT), a gene for a selectable marker and a region of homology to a non-essential region of MP1 or to a non-essential region of CP1, such that the transmissible nonreplicating plasmid integrates into the MP1 or into the CP1 plasmid of D. radiodurans at the region of homology; (b) selecting D. radiodurans for the selectable marker thereby obtaining D. radiodurans transconjugants having the transmissible plasmid integrated into the MP1 or into the CP1 plasmid; (c) isolating DNA from the D. radiodurans selected in (b); and (d) transforming a recipient cell with the isolated DNA, thereby cloning the whole MP1 or the whole CP1 into the recipient cell. In one embodiment of the method of cloning the whole megaplasmid (MP1) or whole small plasmid (CP1) of D. radiodurans, the donor cell includes a non-transmissible plasmid that includes genes necessary for conjugation in trans of the transmissible nonreplicating plasmid. In another embodiment of the method of cloning the whole megaplasmid (MP1) or whole small plasmid (CP1) of D. radiodurans, the transmissible nonreplicating plasmid is a cis- conjugative plasmid having genes for conjugation. In another embodiment of the method of cloning the whole megaplasmid (MP1) or whole small plasmid (CP1) of D. radiodurans, the method further comprises isolating the whole MP1 or the whole CP1 of D. radiodurans from the recipient cell. In another embodiment, the present disclosure provides for a method of creating restriction endonuclease knock-out strains of Deinococcus radiodurans (D. radiodurans), the method comprising: (a) transferring by conjugation a transmissible nonreplicating plasmid from a donor cell to D. radiodurans, the transmissible nonreplicating plasmid including an origin of transfer and a deletion cassette including a gene for a selectable marker between regions of homology flanking or within a restriction endonuclease gene of D. radiodurans such that the selectable marker integrates into or replaces the restriction endonuclease gene of D. radiodurans; and (b) selecting D. radiodurans for the selectable marker, thereby obtaining transconjugants of D. radiodurans that are restriction endonuclease knock-out strains for the restriction endonuclease gene. In one embodiment of the method of creating restriction endonuclease knock-out strains of D. radiodurans, the method further comprises repeating, for a number of times, steps (a) and (b), each time using the knock-out strain obtained in step (b) as the D. radiodurans of step (a), and each time the transmissible nonreplicating plasmid including a different selectable marker from any of the previous times, the different selectable marker being between regions of homology flanking or within a different endonuclease gene of D. radiodurans, wherein the number of times is equal to the total number of restriction endonuclease genes in D. radiodurans minus one, thereby obtaining a restriction minus strain of D. radiodurans. In another embodiment of the method of creating restriction endonuclease knock-out strains of D. radiodurans, the transmissible, non-replicating plasmid is a transmissible, non- replicating cis-conjugative plasmid having genes for conjugation, a gene for a selectable marker, and the DNA fragment of interest. In another embodiment of the method of creating restriction endonuclease knock-out strains of D. radiodurans, the transmissible, non-replicating plasmid is a transmissible, non- replicating plasmid including an origin of transfer (oriT), the gene for the selectable marker, and the DNA fragment of interest, and wherein the donor cell further includes a non-transmissible plasmid that includes genes necessary for conjugation in trans of the transmissible, non-replicating plasmid. In another embodiment of the method of creating restriction endonuclease knock-out strains of D. radiodurans, the DNA molecule is a linear DNA molecule. In another embodiment, the present disclosure relates to a recombinant E. coli including an MP1 megaplasmid, a CP1 small plasmid, a Chr 1 chromosome and/or a Chr 2 chromosome of Deinococcus radiodurans (D. radiodurans). In another embodiment, the present disclosure relates to an isolated or recombinant nucleic acid sequence comprising SEQ ID NO: 1 or an isolated or recombinant nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In another embodiment, the present disclosure relates to a method of cloning a DNA segment from the genome of D. radiodurans into a recipient cell, the method comprising: (a) transferring by conjugation a transmissible plasmid from a first donor cell to D. radiodurans a transmissible plasmid having a gene for a first selectable marker, an origin of transfer (oriT), a counter-selection marker gene disposed between a first region of homology to a first region in the genome of D. radiodurans and a second region of homology to a second region in the genome of D. radiodurans, the first region and the second region flanking the DNA segment, one restriction site between the first region of homology and the counter-selection marker gene and another restriction site between the counter-selection gene and the second region of homology, thereby delivering the transmissible plasmid to D. radiodurans via conjugation; (b) selecting D. radiodurans for the first selectable marker thereby obtaining D. radiodurans transconjugants having the transmissible plasmid; (c) counter selecting colonies of D. radiodurans transconjugants by exposing the D. radiodurans transconjugants of (b) to the counter-selection marker; (d) isolating DNA from the counter selected D. radiodurans transconjugants; and (e) transforming a recipient cell with the isolated DNA, thereby cloning the DNA segment into the recipient cell. In one embodiment of the method of cloning a DNA segment from the genome of D. radiodurans into a recipient cell, the method further comprises isolating the DNA segment from the recipient cell. In another embodiment of the method of cloning a DNA segment from the genome of D. radiodurans into a recipient cell, between steps (a) and (b) the method further comprises exposing the D. radiodurans to a second donor cell that includes genes necessary for conjugation under conditions suitable for conjugation, the second donor cell having a restriction plasmid comprising a restriction endonuclease gene for the one restriction site and the another restriction site, an origin of transfer, an origin of replication and a gene for a second selectable marker, and wherein step (b) comprises selecting the D. radiodurans for the second selectable marker. In another embodiment of the method of cloning a DNA segment from the genome of D. radiodurans into a recipient cell, the counter selectable marker is a visual marker that confers an optical signal to the D. radiodurans transconjugants (b) that retain a copy of the counter selectable marker, or a marker that eliminates D. radiodurans transconjugants (b) that retain a copy of the counter selectable marker in the presence of a counter selective compound. BRIEF DESCRIPTION OF THE DRAWINGS The following figures illustrate various aspects and preferred and alternative embodiments of the present disclosure. Fig. 1. Schematic of a conjugation protocol of DNA transfer to D. radiodurans in accordance with one embodiment. Figs.2A to 2E. Demonstration of pDEINO1 plasmid conjugation from E. coli to D. radiodurans in accordance with one embodiment. (2A) Schematic of the multi-host shuttle plasmid pDEINO1. DrCm ^R , codon-optimized chloramphenicol resistance gene for D. radiodurans. NAT, N-acetyltransferase. NptII, neomycin phosphotransferase II. RepA2B2C2, S. meliloti origin of replication. Pcc1BAC-yeast, backbone for replication in yeast and E. coli. Red lines indicate the location and size (bp) of expected multiplex amplicons and EcoRI-HF cut sites for the diagnostic digest are represented as scissors. (2B) Conjugation frequency of pDEINO1 from E. coli to D. radiodurans with E. coli donors harbouring pTA-Mob and pDEINO1, only pDEINO1, or experiments performed without E. coli donors, only recipient cells. Data are shown as boxplots with points representing individual technical replicates. Error bars represent 95% confidence interval. (2C-2D) Multiplex PCR and diagnostic restriction digest of 30 pDEINO1 plasmids from D. radiodurans transconjugants, following transformation and plasmid induction in E. coli. L, 2-log ladder, +, original pDEINO1 plasmid prior to conjugation. -, water. (2C) Expected multiplex amplicon sizes are 200, 300, 400, and 650 bp. (2D) Expected EcoRI-HF digest band sizes are 3149, 3450, 6548, and 8474 bp. (2E) Phenotypic screening for chloramphenicol sensitivity of D. radiodurans transconjugants harboring pDEINO1 relative to wildtype D. radiodurans by serial dilution spot plates on TGY media and TGY media supplemented with increasing concentration of chloramphenicol. C1-C5, D. radiodurans transconjugant colonies. WT, wildtype D. radiodurans. Figs.3A and 3B. Test of selectable markers on replicating plasmids in D. radiodurans. (3A) Diagram of the selective marker cassettes on pDEINO1, pDEINO3, pDEINO4 and pDEINO5 plasmids containing a codon-optimized cat gene for D. radiodurans coupled with nptII, tetR/A, aadA1 or aacC1, respectively. Created with BioRender.com. (3B) Serial dilutions of D. radiodurans harboring pDEINO1, pDEINO3, pDEINO4 and pDEINO5 alongside wild type D. radiodurans spot plated on TGY media and TGY media supplemented with chloramphenicol, neomycin, tetracycline, streptomycin and gentamicin. Figs.4A to 4C. Demonstration of gene deletions using recombination of conjugative non-replicating plasmids in the D. radiodurans genome in accordance with one embodiment. (4A) Diagram of D. radiodurans genome with the four restriction endonuclease targets indicated as coloured boxes. (B) Conjugative non-replicating plasmid map illustrating the plasmid components of pDEINO7, pDEINO8, pDEINO9 and pDEINO10, used to generate the D. radiodurans restriction endonuclease deletion strains. (C) Multiplex PCR analysis of two D. radiodurans transconjugant colonies for each restriction endonuclease knock out (C1, C2). Controls included wild type D. radiodurans genomic DNA(WT), the original conjugative non- replicating plasmid prior to conjugation (29, 30, 32, 33), and water (-). If present, amplicon sizes are: neomycin 311 bp, tetracycline 409 bp, wild type restriction endonuclease genes ~500 bp, and the non-replicating backbone 645 bp. L, 2-log ladder. Figs.5A to 5E. Cloning of D. radiodurans MP1 megaplasmid in E. coli in accordance with one embodiment. (5A) Schematic of the pDEINO2 nonreplicating plasmid. Multiplex amplicon positions are indicated as red lines with the number indicating the amplicon size. (5B) Diagram illustrating conjugation of the pDEINO2 plasmid from E. coli to D. radiodurans. (5C) Diagram illustrating integration of pDEINO2 into the MP1 plasmid in D. radiodurans through recombination at the McrC gene. Multiplex amplicon positions are indicated as red lines with the number indicating the amplicon size. (5D) Agarose gel of MfeI-HF and NheI-HF restriction digest of three cloned MP1 plasmids recovered from E. coli clones following induction. Expected visible band sizes (<10 kb) for the MfeI-HF digest are 325, 3186, 4338, 4929, 6170, 7049, 7733, and 8948 bp. Expected visible band sizes (<10 kb) for the NheI-HF digest are 2701, 3139, 5070, and 8117 bp. (5E) Agarose gel of multiplex PCR of three cloned MP1 plasmids recovered from E. coli amplifying 200, 300 and 400 bp amplicons from the integrated pDEINO2 plasmid and 200, 300, 400 and 600 bp amplicons from the MP1 plasmid. L, 2-log ladder. +, D. radiodurans wild type genomic DNA. -, water. Fig.6. Test of kanamycin and neomycin sensitivity of D. radiodurans harboring pDEINO1 compared to wild type. Fig. 7. Multiplex PCR for the pDEINO2 plasmid amplified from the DNA of three D. radiodurans transconjugant colonies following conjugation to clone the MP1 megaplasmid. Figs. 8A to 8D. (8A) Diagram of steps in the development of a seamless gene deletion method (SD strategy) in Deinococcus radiodurans in accordance with one embodiment: STEP 1: The SD cassette contains nptII and lacZ genes for antibiotic selection and visual screening following transformation of the SD cassette into D. radiodurans. Homologous recombination of the 1 kb H1 and H2 regions occurs with the D. radiodurans genome resulting in integration of the SD cassette into the genome replacing the GOI. STEP 2: The pSLICER plasmid is conjugated into D. radiodurans where it expresses an I-SceI homing endonuclease that cuts at the 18-bp I-SceI restriction site within the SD cassette. This double-strand break triggers homologous recombination between the duplicated 3’ 80 bp of H1, removing the nptII and lacZ markers. STEP 3: Finally, plasmid curing to remove the pSLICER plasmid results in a marker-free D. radiodurans ΔGOI strain. SD, seamless deletion; H1, homology region 1; H2, homology region 2; GOI, gene of interest; nptII, neomycin resistance gene; lacZ, β-galactosidase. (8B) Schematic of a representative seamless deletion plasmid (pSD) with the general components contained on pSD1, pSD2, pSD3 and pSD4: H1, homology region 1; I-SceI site, I-SceI endonuclease recognition site; Nm ^R , neomycin resistance gene (nptII); lacZ, β-galactosidase gene; 3’ 80 bp of homology 1, duplication of the last 80 bp of H1; H2, homology region 2; oriT, origin of transfer; pCC1BAC- yeast, backbone for selection and replication in yeast and E. coli; Strep ^R , streptomycin resistance gene (aadA1); Tet ^R , tetracycline resistance gene (tetR/A). The SD cassette is indicated with a dotted line. (8C) Schematic of a pSLICER: Origin of replication, origin of replication for D. radiodurans; drCm ^R , chloramphenicol resistance gene codon-optimized for D. radiodurans; drI-SceI, I-SceI endonuclease codon-optimized for D. radiodurans. Figs. 9A to 9C. Seamless deletion of RM1-4 genes in D. radiodurans in accordance with one embodiment. (9A) Representative schematic of the multiplex PCR amplicons present in D. radiodurans strains: wild type, with the SD cassette integrated at the RM2 locus, with the addition of the SLICER plasmid seamlessly removing the SD cassette, and the final D. radiodurans ΔRM2 strain. Amplicons are shown as green lines with the corresponding size in bp written on top. Created with BioRender.com. (9B) Spot plates of 10-fold serial dilutions of the same strains listed in (A). All plates contain X-Gal 40 μg mL ^-1 . (9C) Gel electrophoresis of multiplex PCR results from all steps in the creation of the four seamless R-M gene deletions. The PCR for each deletion (RM1-RM4) was performed on DNA from a single D. radiodurans colony in the order depicted in (A): D. radiodurans gDNA (WT), following integration of the SD cassette (+SD), following conjugation of the SLICER plasmid (+SLI), and following plasmid curing (ΔRM1, ΔRM1-2, ΔRM1-3, and ΔRM1-4). Controls include the SD plasmid DNA extracted from E. coli (SD) and wild-type D. radiodurans gDNA (WT). For the RM1 multiplex, a wild-type (WT) genomic DNA control is used, and for all subsequent multiplex analyses the cured strain from the previous deletion was used as a control (ΔRM1, ΔRM1-2, ΔRM1-3). Expected amplicon sizes are approximately 150 bp for the D. radiodurans gDNA control, 300 bp for nptII in the SD cassette, 500 bp for the R-M gene, and 650 bp for the SLICER plasmid backbone. L, 1 kb plus ladder. Figs.10A and 10B. D. radiodurans ΔRM1-4 multiplex PCR analysis. (10A) Schematic representation of the D. radiodurans ΔRM1-5 Nm ^R genome with the first four R-M genes deleted (RM1, RM2, RM3, RM4) as indicated by grey X’s, and the fifth R-M system (RM5) replaced with a neomycin marker (Nm ^R ) as indicated in pink. Created with BioRender.com. (10B) Gel electrophoresis of multiplex PCR performed on DNA extracted from a single D. radiodurans ΔRM1-4 colony for each of the four seamless R-M gene deletions (RM1-4). For the RM1 multiplex, a wild-type (WT) genomic DNA control is used, and for all subsequent multiplex analyses the cured strain from the previous deletion was used as a control (ΔRM1, ΔRM1-2, ΔRM1-3). A negative control (-) where water was used in place of template was also included. Expected amplicon sizes are approximately 150 bp for the D. radiodurans gDNA control and 500 bp for the R-M gene, if present. L, 1 kb plus ladder. Fig.11A and 11B. Physiological analysis of D. radiodurans seamless deletion strains. (11A) Growth curves of D. radiodurans WT and ΔRM knockout strains grown in liquid TGY media for 17 hours. Each data point represents the mean of three biological and two technical replicates, with error bars representing standard error of the mean. The average doubling time for each strain is reported in the legend in minutes and represents the mean value of three biological and two technical replicates ± the standard deviation. (11B) Number of D. radiodurans transformants following heat shock transformation of the pRAD1 plasmid to D. radiodurans WT and ΔRM knockout strains.850 ng of DNA was used for each transformation and the number of D. radiodurans transformants that grew on TGY supplemented with chloramphenicol 3 µg mL ^-1 is reported. Data is presented as a bar graph indicating the mean of three biological replicates with error bars representing standard error of the mean. Fig.12. Selective plates following conjugation of pDEINO1 and pSLICER from E. coli to D. radiodurans ΔRM1-4 Nm ^R . Antibiotic concentrations are reported as µg mL ^-1 . Fig.13. Step-by-step SLICER protocol. Laboratory protocol for the SLICER method in accordance with one embodiment of the present disclosure, which can be used to create a seamless gene deletion in D. radiodurans in approximately 2 weeks. DESCRIPTION Definitions The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology. All numerical designations, e.g., pH, temperature, time, concentration and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 1.0 or 0.1, as appropriate, or alternatively by a variation of +/- 15 %, or alternatively 10%, or alternatively 5% or alternatively 2%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a polypeptide” includes a plurality of polypeptides, including mixtures thereof. As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the intended use. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure. As used herein “contacting” means a method to deliver a conjugative plasmid to a Deinococcus radiodurans cell using conjugative molecular biological techniques of the present disclosure. The plasmid can be delivered as an isolated DNA or isolated plasmid, or it can be delivered within a system by being carried in another bacterium, bacteriophage, a liposome or any other cell delivery system. The plasmid may also be delivered naked. A “subject” of treatment is a cell or an animal such as a mammal or a human. Non-human animals subject to treatment and are those subject to infections or animal models, for example, simians, murines, such as, rats, mice, chinchilla, canine, such as dogs, leporids, such as rabbits, livestock, sport animals and pets. Non-animal subjects of treatment would include as non- exclusive examples bioreactors, treatment plants, landfills etc. The term “isolated” or “recombinant” as used herein with respect to nucleic acids, such as DNA or RNA, or plasmids refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule as well as polypeptides. The term “isolated or recombinant plasmids” is meant to include plasmids which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polynucleotides, polypeptides and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated or recombinant” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype. An isolated polynucleotide is separated from the 3’ and 5’ contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. As used herein, the terms “treating,” “treatment” and the like are used herein to mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder. To “prevent” intends to prevent a disorder or effect in vitro or in vivo in a system or subject that is predisposed to the disorder or effect. An example of such is preventing the formation of a biofilm in a system that is infected with a microorganism known to produce one. “Pharmaceutically acceptable carriers” refers to any diluents, excipients or carriers that may be used in the compositions of the present disclosure. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They are preferably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like and consistent with conventional pharmaceutical practices. “Administration” can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated and target cell or tissue. Non-limiting examples of route of administration include oral administration, nasal administration, injection and topical application. “DNA fragment” refers to a fraction of a deoxyribonucleic acid (DNA) molecule. “DNA fragment” can include a gene, a restriction endonuclease gene, a promoter, and so forth. A “DNA segment” refers to a nucleotide sequence within a DNA molecule. “Plasmid” refers to an extra-chromosomal DNA molecule separate from the chromosomal DNA. Plasmids replicate extra-chromosomally inside a cell and can transfer their DNA from one cell to another by a variety of mechanisms. DNA sequences controlling extra chromosomal replication (ori) and transfer (tra) are distinct from one another; i.e., a replication sequence generally does not control plasmid transfer, or vice-versa. A “conjugative plasmid” is a plasmid that is transferred from one organism, such as a bacterial cell, to another organism during a process termed conjugation. The term refers to a self- transmissible plasmid that carries genes promoting the plasmid’s own transfer by conjugation. Cis- conjugative plasmids carry their own origin of replication, oriV, and an origin of transfer, oriT, and genes promoting the plasmid’s own transfer by the conjugation process. When conjugation is initiated, a relaxase enzyme creates a “nick” in one plasmid DNA strand at the oriT. The enzyme may work alone or in a complex of over a dozen proteins. The transferred, or T-strand, is unwound from the plasmid and transferred into the recipient bacterium in a 5′-terminus to 3′-terminus direction through a conjugative pilus. The remaining strand is replicated, either independent of conjugative action (vegetative replication, beginning at the oriV) or in concert with conjugative replication. Conjugation functions can be plasmid encoded, but some conjugation genes can be found in the bacterial chromosome or another plasmid and can exhibit their activity in trans to a separate plasmid that encodes the oriT sequence. Numerous conjugative plasmids are known, which can transfer associated genes within one species (narrow host range) or between many species (broad host range). Conjugation can occur between species classified as different at any taxonomic level---including in the extreme between domains, e.g., bacteria to eukaryotes. “Origin of transfer” or “oriT” refers to the cis-acting site required for DNA transfer. Integration of an oriT sequence into a non-transmissible plasmid converts said plasmid into a transmissible or mobilizable plasmid. A cis-conjugative plasmid is a plasmid that encodes both the conjugative machinery and a gene or combination of genes for targeted bacterial modulation, including killing of bacteria (such as CRISPR nuclease), metabolic manipulation of bacteria and augmentation of beneficial bacteria, as well as for the detection of bacteria and so forth. The term “effective amount” refers to a quantity sufficient to achieve a beneficial or desired result or effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical compositions. In some embodiments the effective amount is the amount sufficient to result in a protective response against a pathogen. In other embodiments, the effective amount of an immunogenic composition is the amount sufficient to result in antibody generation against an antigen. In some embodiments, the effective amount is the amount required to confer passive immunity on a subject in need thereof. With respect to immunogenic compositions, in some embodiments the effective amount will depend on the intended use, the degree of immunogenicity of a particular antigenic compound, and the health/responsiveness of the subject’s immune system, in addition to the factors described above. The skilled artisan will be able to determine appropriate amounts depending on these and other factors. “Seamless deletion” or “scarless deletion” is used in this document to refer to the deletion of a nucleotide fragment from a nucleotide sequence that includes the nucleotide fragment without leaving an exogenous or foreign nucleotide segment in the nucleotide sequence. In one embodiment, the term is also used to refer to a marker-less insertion of a gene of interest. In one embodiment, the seamless deletion of a gene includes also inserting a gene of interest into the nucleotide sequence. In the case of an in vitro application, in some embodiments the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target and the methods in use. The skilled artisan will be able to determine the effective amount based on these and other considerations. The effective amount may comprise one or more administrations of a composition depending on the embodiment. The agents and compositions can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions. The terms “equivalent” or “biological equivalent” are used interchangeably when referring to a particular molecule, biological, or cellular material and intend those having minimal homology while still maintaining desired structure or functionality. It is to be inferred without explicit recitation and unless otherwise intended, that when the present disclosure relates to a plasmid, polypeptide, protein, or polynucleotide, an equivalent or a biologically equivalent of such is intended within the scope of this disclosure. As used herein, the term “biological equivalent thereof” is intended to be synonymous with “equivalent thereof” when referring to a reference protein, antibody, polypeptide or nucleic acid or plasmid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. For example, an equivalent intends at least about 70 % homology or identity, or alternatively about 80 % homology or identity and alternatively, at least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 % or alternatively 98 % percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid. In another aspect, the term intends a polynucleotide that hybridizes under conditions of high stringency to the reference polynucleotide or its complement. A polynucleotide or polynucleotide sequence (or a polypeptide or polypeptide sequence) having a certain percentage (for example, 80%, 85%, 90% or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds.1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST. “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non- homologous” sequence shares less than 30% identity or alternatively less than 25% identity, less than 20 % identity, or alternatively less than 10% identity with one of the sequences of the present disclosure. “Homology” or “identity” or “similarity” can also refer to two nucleic acid molecules that hybridize under stringent conditions to the reference polynucleotide or its complement. Overview Provided herein are novel strains of D. radiodurans, methods of transfer of a nucleotide sequence from a donor cell to D. radiodurans and methods of deleting target nucleotide sequences from D. radiodurans’ genome. The methods of the present disclosure enable the deletion, including seamless deletions, of target nucleotide fragments in the genome of D. radiodurans, the construction of D. radiodurans strains that are restriction minus (i.e., that lack any and/or all restriction endonuclease sites), and the cloning of whole chromosome or plasmid of D. radiodurans’ genome. The present disclosure relates also to modified pAGE3.0 plasmids [[31]] with a gene for a selectable marker (such as chloramphenicol, neomycin, tetracycline, streptomycin or gentamycin, a visual marker or a supplemented marker; other antibiotic markers include nptII (KanR), aadA (SpecR), cat (CmR) and aac(3) (GenR)) with or without an origin of replication for D. radiodurans (Figs. 2A and 5A). This plasmid, like all pAGE plasmids, permits replication and selection in a number of cells, including E. coli, S. cerevisiae, S. meliloti and now D. radiodurans. The plasmid of the present disclosure also has an origin of transfer necessary for direct DNA transfer via conjugation. Conjugation has proven to be a versatile and simple method of DNA transfer capable of moving large plasmids to bacteria and eukaryotic organisms (Fig.2). DNA delivery method In one embodiment, a method for delivering a DNA fragment to Deinococcus radiodurans (D. radiodurans), includes: (a) transferring by conjugation a transmissible plasmid from a donor cell to D. radiodurans, the transmissible plasmid including the DNA fragment of interest and a selectable marker; and (b) selecting D. radiodurans for the selectable marker, wherein the selected D. radiodurans include the DNA fragment of interest. In one embodiment, step (a) includes exposing D. radiodurans to a donor cell under conditions suitable for conjugation. In one embodiment, the transmissible plasmid is a transmissible cis-conjugative plasmid having genes for conjugation, a gene for a selectable marker, and the DNA fragment of interest. In another embodiment, the transmissible plasmid includes an origin of transfer (oriT), the gene for the selectable marker, and the DNA fragment of interest, and wherein the donor cell includes a non-transmissible plasmid that includes genes necessary for conjugation in trans of the transmissible plasmid. In one embodiment, the transmissible plasmid can be non-replicating or replicating. The replicating transmissible plasmid includes an origin of replication for D. radiodurans. In one embodiment, the replicating plasmid includes a selectable marker and origin of replication for D. radiodurans to create pDEINO1 (Fig.2A). Conjugation is then performed using a donor cell such as E. coli ΔdapA donor strain harbouring pTA-Mob (https://doi.org/10.1371/journal.pone.0090372), an IncP RK2 conjugative plasmid encoding all the genes necessary for conjugation, and the ~22 kb cargo plasmid, pDEINO1. The oriT on the cargo plasmid is recognized and nicked by relaxase expressed from pTA-Mob and is shuttled to the D. radiodurans recipient strain. Conjugation from E. coli to D. radiodurans was observed with an average conjugation frequency of 1.12 x 10 ^-5 transconjugants per recipient (Fig.2B), while no transconjugants were observed using donors carrying pDEINO1 mixed with recipient, or with recipient alone. In another embodiment, the DNA transfer to D. radiodurans, conjugation is achieved using a donor cell harboring a replicating cis-conjugative plasmid having genes for conjugation, a gene for a selectable marker, an origin of replication for D. radiodurans and the DNA fragment to be transferred to D. radiodurans. Other host cells that can be used in the different embodiments of the present disclosure include Sinorhizobium meliloti, E. coli, including E. coli with different deletions (including different auxotrophic strains). Whole chromosome cloning and engineering In another embodiment, the present disclosure provides for a method of cloning whole chromosome or plasmid (MP1 or CP1) of D. radiodurans’ genome comprising: (a) transferring by conjugation a transmissible nonreplicating plasmid from a donor cell to D. radiodurans, the transmissible plasmid including an origin of transfer (oriT), a gene for a selectable marker and a region of homology to a non-essential region of MP1 or to a non-essential region of CP1, such that the transmissible nonreplicating plasmid integrates into the MP1 or into the CP1 plasmid of D. radiodurans at the region of homology; (b) selecting D. radiodurans for the selectable marker thereby obtaining D. radiodurans transconjugants having the transmissible plasmid integrated into the MP1 or into the CP1 plasmid; (c) isolating DNA from the D. radiodurans selected in (b); and (d) transforming a recipient cell with the isolated DNA, thereby cloning the whole MP1 or the whole CP1 into the recipient cell. In one embodiment, the donor cell includes a non-transmissible plasmid that includes genes necessary for conjugation in trans of the transmissible nonreplicating plasmid. In another embodiment, the transmissible nonreplicating plasmid is a cis-conjugative plasmid having genes for conjugation. In embodiments, the method further comprises isolating the whole MP1 or the whole CP1 of D. radiodurans from the recipient cell. In one embodiment, native CP1 and MP1 plasmids of D. radiodurans, which are approximately 60 kb and 200 kb, respectively, have been cloned in vivo through homologous recombination. Nonreplicating plasmids (pDEINO-CP1-IN and pDEINO-MP1-IN) for D. radiodurans were synthesized containing elements for selection and replication in E. coli and/or yeast, a selectable marker for D. radiodurans, an origin of transfer, and a 1 kb region of homology to the respective target native chromosome or native plasmid. The synthesized nonreplicating plasmids were transformed into a conjugative donor cell and delivered to D. radiodurans via the conjugation method of the present disclosure. Using MP1 as an example, transconjugants that appeared on the MP1 selective plates were grown overnight, DNA was extracted and a multiplex PCR for the pDEINO-MP1-IN plasmid was performed. This extracted DNA was then transformed into a donor cell and colonies were screened by multiplex PCR for the pDEINO-MP1-IN plasmid and the D. radiodurans MP1 plasmid as well as diagnostic digests. Cloning of Genome Fragments In another embodiment, the present disclosure provides for a method of cloning a segment of the D. radiodurans’ genome. In one embodiment, a method of cloning a DNA segment from the genome of Deinococcus radiodurans (D. radiodurans) into a recipient cell comprises: (a) transferring by conjugation a transmissible plasmid from a first donor cell to D. radiodurans a transmissible plasmid having a gene for a first selectable marker, an origin of transfer (oriT), a counter-selection marker gene disposed between a first region of homology to a first region in the genome of D. radiodurans and a second region of homology to a second region in the genome of D. radiodurans, the first region and the second region flanking the DNA segment, one restriction site between the first region of homology and the counter-selection marker gene and another restriction site between the counter- selection gene and the second region of homology, thereby delivering the transmissible plasmid to D. radiodurans via conjugation; (b) selecting D. radiodurans for the first selectable marker thereby obtaining D. radiodurans transconjugants having the transmissible plasmid; (c) counter selecting colonies of D. radiodurans transconjugants by exposing the D. radiodurans transconjugants of (b) to the counter-selection marker; (d) isolating DNA from the counter selected D. radiodurans transconjugants; and (e) transforming a recipient cell with the isolated DNA, thereby cloning the DNA segment into the recipient cell. In one embodiment, the method further comprises isolating the DNA segment from the recipient cell. In another embodiment, between steps (a) and (b) the method further comprises exposing the D. radiodurans to a second donor cell that includes genes necessary for conjugation under conditions suitable for conjugation, the second donor cell having a restriction plasmid comprising a restriction endonuclease gene for the one restriction site and the another restriction site, an origin of transfer, an origin of replication and a gene for a second selectable marker, and wherein step (b) comprises selecting the D. radiodurans for the second selectable marker. In one embodiment, the counter selectable marker is a visual marker that confers an optical signal to the D. radiodurans transconjugants (b) that retain a copy of the counter selectable marker, or a marker that eliminates D. radiodurans transconjugants (b) that retain a copy of the counter selectable marker in the presence of a counter selective compound. The counter selectable marker can be a visual marker that confers an optical signal to the D. radiodurans transconjugants of (b) that retain a copy of the counter selectable marker. For example, the counter selection marker can be GFP (green fluorescent protein) and with proper integration the plasmid the D. radiodurans cells that have the plasmid integrated in the desired position will stop being fluorescent. The counter selection marker can also be a compound that eliminates D. radiodurans transconjugants of (b) that retain a copy of the counter selectable marker in the presence of said compound. Restriction minus strain of D. radiodurans The present disclosure presents also embodiments to a method for deleting one or more restriction endonuclease genes, including all restriction endonuclease genes, from the genome of D. radiodurans. The method of creating restriction endonuclease knock-out strains of D. radiodurans comprises: (a) transferring by conjugation a transmissible nonreplicating plasmid from a donor cell to D. radiodurans, the transmissible nonreplicating plasmid including an origin of transfer and a deletion cassette including a gene for a selectable marker between regions of homology flanking or within a restriction endonuclease gene of D. radiodurans such that the selectable marker integrates into or replaces the restriction endonuclease gene of D. radiodurans; and (b) selecting D. radiodurans for the selectable marker, thereby obtaining transconjugants of D. radiodurans that are restriction endonuclease knock-out strains for the restriction endonuclease gene. In one embodiment, the method further comprises repeating, for a number of times, steps (a) and (b), each time using the knock-out strain obtained in step (b) as the D. radiodurans of step (a), and each time the transmissible nonreplicating plasmid including a different selectable marker from any of the previous times, the different selectable marker being between regions of homology flanking or within a different endonuclease gene of D. radiodurans, wherein the number of times is equal to the total number of restriction endonuclease genes in D. radiodurans minus one, thereby obtaining a restriction minus strain of D. radiodurans. In one embodiment, each time, the different selectable marker is located between regions of homology flanking or within a different endonuclease gene of D. radiodurans. In embodiments, the number of times is equal to the total number of restriction endonuclease genes in D. radiodurans minus one (because the first restriction endonuclease gene was removed in the first pass), thereby obtaining a restriction minus strain of D. radiodurans. That is, steps (a) and (b) are repeated as described herein for as many restriction endonuclease genes are left in the genome. In one embodiment, the transmissible, non-replicating plasmid is a transmissible, non- replicating cis-conjugative plasmid having genes for conjugation, a gene for a selectable marker, and the DNA fragment of interest. In another embodiment, the transmissible, non-replicating plasmid is a transmissible, non- replicating plasmid including an origin of transfer (oriT), the gene for the selectable marker, and the DNA fragment of interest, and wherein the donor cell further includes a non-transmissible plasmid that includes genes necessary for conjugation in trans of the transmissible, non-replicating plasmid. In another embodiment, the DNA molecule is a linear DNA molecule. There are at least 6 predicted genes in the D. radiodurans genome that encode for restriction endonucleases. Four restriction endonuclease targets in the D. radiodurans genome, their location and characterization are summarized in Fig. 4A. Plasmids have been designed to include a selectable marker. The restriction minus strain of D. radiodurans is an important technological advance for the use of D. radiodurans as a tool in DNA assembly. Cell extracts and so forth. Gene Deletion The present disclosure, in another embodiment, provides for a method of deleting a target gene of D. radiodurans and creating knockout strains of D. radiodurans. The method, in one embodiment, comprises: (a) exposing D. radiodurans to a donor cell under conditions suitable for conjugation, the donor cell having either (i) a transmissible non replicating plasmid having an origin of transfer and a deletion cassette including a gene for a selectable marker between regions of homology flanking or within the target gene of D. radiodurans and a non-transmissible plasmid that includes genes necessary for trans-conjugation, or (ii) a non-replicating cis-conjugative plasmid having genes for conjugation, and the deletion cassette, thereby delivering the plasmid of (i) or (ii) to the D. radiodurans via conjugation such that the selectable marker integrates into the target gene; and (b) selecting D. radiodurans for the selectable marker, thereby obtaining transconjugants of D. radiodurans that are knock-out strains for the target gene. Seamless Gene Deletion In another embodiment, the present disclosure is a method for seamless deletion of a target DNA sequence in the genome of D. radiodurans as illustrated in Figs.8A to 8D. In embodiments, the target DNA sequence in D. radiodurans is a target gene or gene of interest (GOI), including essential GOIs and nonessential GOIs. In one embodiment, a method of seamless deletion of a target nucleotide fragment of D. radiodurans comprises: (a) transferring a DNA molecule to D. radiodurans, the DNA molecule having a seamless deletion (SD) cassette including in a 5’ to 3’ order (A) a first homology region to a first region in the genome of D. radiodurans, (B) a restriction endonuclease site that is not present in D. radiodurans (unique restriction endonuclease site), (C) a gene for a first selectable marker, (D) another homology region to the first region in the genome of D. radiodurans, the another homology region having less nucleotides than the first homology region, and (E) a second homology region to a second region in the genome of D. radiodurans, wherein the target nucleotide fragment is between the first region in the genome of D. radiodurans and the second region in the genome of D. radiodurans; (b) selecting D. radiodurans for the selectable marker, thereby obtaining D. radiodurans that include the SD cassette and deletion of the target nucleotide fragment; and (c) transferring a replicating plasmid to the D. radiodurans, the replicating plasmid including (i) a second selectable marker that is different from the first selectable marker and (ii) a gene encoding a restriction endonuclease that recognizes the unique restriction endonuclease site, whereby expression of the restriction endonuclease in the selected D. radiodurans cuts the unique restriction endonuclease site to obtain transconjugants of D. radiodurans with seamless deletion of the target nucleotide fragment. In one embodiment of the method of seamless deletion of a target nucleotide fragment of D. radiodurans, the DNA molecule is a transmissible, non-replicating plasmid, and wherein step (a) comprises transferring the transmissible, non-replicating plasmid by conjugation from a donor cell to D. radiodurans. In one embodiment of the method of seamless deletion of a target nucleotide fragment of D. radiodurans, the transmissible, non-replicating plasmid is a transmissible, non-replicating cis- conjugative plasmid having genes for conjugation. In one embodiment of the method of seamless deletion of a target nucleotide fragment of D. radiodurans, the transmissible, non-replicating plasmid is a transmissible, non-replicating plasmid including an origin of transfer (oriT), and wherein the donor cell further includes a non- transmissible plasmid that includes genes necessary for conjugation in trans of the transmissible, non-replicating plasmid. In embodiments, the donor cell is E. coli or Sinorhizobium meliloti. In one embodiment of the method of seamless deletion of a target nucleotide fragment of D. radiodurans, the DNA molecule is a linear DNA molecule. In one embodiment of the method of seamless deletion of a target nucleotide fragment of D. radiodurans, the SD cassette further includes a gene of interest between the another homology region and the second homology region, and wherein the transconjugants of D. radiodurans with seamless deletion of the target nucleotide fragment includes the gene of interest. In one embodiment of the method of seamless deletion of a target nucleotide fragment of D. radiodurans, the gene for the first selectable marker and the gene for the second selectable marker confer D. radiodurans resistance to the first selectable marker and to the second selectable marker. In embodiments, the gene for the first selectable marker comprises at least two genes for selectable markers: one gene for selection (for example a gene that confers antibiotic resistance) and another one for screening (for example, lacZ (β-galactosidase) marker). In one embodiment of the method of seamless deletion of a target nucleotide fragment of D. radiodurans, the gene for the first selectable marker comprises: (i) an antibiotic resistance gene for selection, and (ii) a gene for a screening marker that confers an optical signal to the D. radiodurans having the SD cassette. In one embodiment of the method of seamless deletion of a target nucleotide fragment of D. radiodurans, the target nucleotide fragment is a gene, a restriction endonuclease site, a promoter, or an intragenic region. In one embodiment of the method of seamless deletion of a target nucleotide fragment of D. radiodurans, the target nucleotide fragment is at least one restriction enzyme gene. In one embodiment of the seamless GOI deletion, a DNA molecule that includes seamless deletion (SD) cassette that targets the gene of interest is provided. With reference to Fig.8A, the SD cassette includes a marker for selection and screening in D. radiodurans flanked by two regions homologous to the D. radiodurans genome. These two homology regions (also referred to as first homology region and second homology region) are the sequences upstream and downstream of the target DNA sequence (i.e., the GOI). Following the first homology region, there is a unique endonuclease recognition site and prior to the second homology region there is a segment of the first homology region (in one embodiment, a duplication of the last about 80 bp of the first homology region). In one embodiment, a second selective marker for D. radiodurans is located on the DNA molecule outside of the SD cassette. The unique endonuclease recognition site is a recognition site that is not found in wild-type genome of D. radiodurans. In Fig. 8A, the recognition site is an 18-bp I-SceI endonuclease recognition site. There are no I-Scel recognition sites in wild-type D. radiodurans. In one embodiment, first step in the seamless gene deletion method is the integration of the SD cassette into the D. radiodurans genome at the target locus (i.e., at the GOI). In one embodiment, the SD cassette, which is a linear DNA molecule, is delivered to D. radiodurans by chemical transformation. In another embodiment, the SD cassette is inserted into a nonreplicating plasmid. The whole nonreplicating plasmid carrying the SD cassette is then delivered to D. radiodurans via conjugation. Next homologous recombination of the first and second homology regions with the corresponding genomic regions in D. radiodurans results in integration of the SD cassette into the D. radiodurans genome, replacing the GOI. D. radiodurans transformants containing the cassette can be selected on media supplemented with the marker for selection. The resulting strain is referred to as D. radiodurans + SD. A second step involves the removal of the SD cassette. A cassette having a restriction enzyme gene that recognizes the unique recognition site (for example I-SceI endonuclease) and a selectable marker that is different from the selectable marker in the SD cassette is then delivered on a replicative plasmid by conjugation. Expression of the endonuclease results in a cut at the unique recognition site followed by homologous recombination between the first homology region and the about 80-bp duplicated region. At the end of the second step the marker included in the SD cassette is lost due to the homologous recombination. The resulting strain is referred to as D. radiodurans + replicative plasmid. A third step is to cure the D. radiodurans strain obtained in the second step of the replicative plasmid. The D. radiodurans strain obtained in the second step is propagated on nonselective media. Single colonies are then grown on nonselective media and on selective media. The colonies are confirmed to be cured of the replicative plasmid when growth is observed on nonselective plates but not on selective plates. Using the seamless deletion strategy of the present disclosure, genetically modified strains of D. radiodurans have been created in which target genes have been selectively knocked out or deleted (see non limiting examples of Figs.9A and 9B). In one embodiment, the present disclosure provides for genetically modified D. radiodurans strains having at least one restriction enzyme gene knocked out. In one embodiment, the genetically modified D. radiodurans comprise at least two, or at least three, or at least four, or at least five, or at least six restriction enzyme genes knocked out. In another embodiment, the genetically modified D. radiodurans comprise all restriction enzyme genes of wild-type D. radiodurans being knocked out. D. radiodurans restriction enzyme genes include Mrr, Mrr2, ORF2230, ORF14075, ORF15360 and McrBC. In embodiments, the present disclosure provides also for (a) genetically modified D. radiodurans strains without any one of the MP1 megaplasmid, the CP1 small plasmid, the DR_Main chromosome and/or the DR412 chromosome of wild-type D. radiodurans; (b) genetically modified D. radiodurans strains without two, three or all four of the MP1 megaplasmid, the CP1 small plasmid, the DR_Main chromosome and the DR412 chromosome, and (c) genetically modified D. radiodurans strains having one or more deleted genes or DNA fragments. In embodiments, cell extracts can be obtained from any of the produced D. radiodurans strains of the present disclosure. The present disclosure provides also for non-D. radiodurans cells that include a piece of D. radiodurans’ genome. For example, an E. coli strain including MP1 megaplasmid, CP1 small plasmid, DR_Main chromosome and/or DR412 chromosome. In embodiments, the present disclosure provides also for an isolated or recombinant nucleic acid sequence comprising SEQ ID NO: 1 or an isolated or recombinant nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In embodiments, the present disclosure relates also to an isolated functional fragment of SEQ ID NO: 1. In embodiments, the present disclosure relates also to a kit comprising: (a) an isolated conjugative plasmid comprising conjugation genes and a gene or a combination of genes capable of being expressed in D. radiodurans that modulates the D. radiodurans; and (b) instructions for conjugating the conjugative plasmid into D. radiodurans. EXAMPLES These Examples are described solely for purposes of illustration and are not intended to limit the scope of the disclosure. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Example 1 Materials and Methods Microbial Strains and Growth Conditions. Deinococcus radiodurans R1 (acquired from Dr. Murray Junop at Western University) was grown in TGY medium (5 g L ^-1 tryptone, 3 g L ^-1 yeast extract, 1 g L ^-1 potassium phosphate dibasic, and 2.5 mL 40% w/v glucose) supplemented with appropriate antibiotics (chloramphenicol 5 μg mL ^-1 , neomycin 5 ug mL ^-1 , tetracycline 0.5 ug mL ^-1 ) at 30°C. Escherichia coli (Epi300, Lucigen, Cat #: EC300110) was grown at 37°C in Luria Broth (LB) supplemented with appropriate antibiotics (chloramphenicol 15 μg mL ^-1 ). Escherichia coli ECGE101 (ΔdapA) [31] was grown at 37°C in LB supplemented with diaminopimelic acid (DAP) 60 μg mL ^-1 and appropriate antibiotics (chloramphenicol 15 μg mL ^-1 ) and gentamicin 40 μg mL ^-1 ). Saccharomyces cerevisiae VL6−48 (ATCC MYA-3666: MATα his3-Δ200 trp1-Δ1 ura3−52 lys2 ade2−1 met14 cir ⁰ ) was grown at 30°C in 2X YPAD rich medium (20 g L ^-1 yeast extract, 40 g L ^-1 peptone, 40 g L ^-1 glucose, and 200 mg L ^-1 adenine hemisulfate) supplemented with 80 mg L ^-1 adenine hemisulfate, or in complete minimal medium lacking histidine supplemented with 60 mg L ^-1 adenine sulfate (Teknova, Inc., Cat #: C7112) with 1 M sorbitol. A list of strains used in this study is provided in Table 2. Plasmid Design and Construction. All plasmids in this study (Table 3) were constructed from PCR amplified DNA fragments assembled using a yeast spheroplast transformation protocol as previously described [37]. The primers used to amplify the fragments for assembly contained 20 bp binding and 40 bp of overlapping homology to the adjacent DNA fragment. A list of assembly primers for each plasmid is provided in Table 4. Following assembly, DNA was isolated from the S. cerevisiae and the plasmid pool was electroporated into E. coli Epi300. Plasmids from individual colonies were screened for correct assembly using multiplex PCR and a diagnostic digest, and one final clone was confirmed by next-generation sequencing (MGH CCIB DNA Core, Massachusetts, USA). All plasmids permit replication and selection in E. coli and S. cerevisiae and have a low-copy E. coli origin that can be induced to high copy with arabinose. They also have an origin of transfer (oriT) necessary for direct DNA transfer via conjugation. The pDEINO1 plasmid was constructed by restriction digestion of pAGE3.0 [31] with PacI to insert a synthesized codon-optimized chloramphenicol gene under the control of a constitutive promoter (drKatA) and an origin of replication for D. radiodurans from pRadDEST-GFP and pRAD1 [16], respectively. This plasmid also allows replication and selection in other alternative host organisms, including S. meliloti and P. tricornutum, and contains a neomycin marker for S. meliloti that is functional in D. radiodurans as well. The pDEINO20, pDEINO37 and pDEINO39 plasmids contain an origin of replication for D. radiodurans from pRadDEST-GFP, and a tetracycline, streptomycin and gentamicin gene, respectively. Conjugation from E. coli to D. radiodurans. The E. coli ECGE101 ΔdapA donor strain [31], harboring pTA-Mob [30] and pDEINO1, and a control strain, harboring only pDEINO1, were grown at 37°C overnight in 5 mL of LB media supplemented with diaminopimelic acid 60 μg mL- ¹ , gentamicin 40 μg mL ^-1 (donor only), and chloramphenicol 15 μg ^-1 . The saturated E. coli cultures were diluted 1:50 into 50 mL of the same media and grown for ~2 h to an OD ₆₀₀ of 0.6. The D. radiodurans R1 recipient strain was grown at 30°C overnight in TGY media to an OD ₆₀₀ of 0.6. Donor, control, and recipient cultures were transferred to 50 mL falcon tubes and centrifuged at 5,000 x g for 10 min at 4°C. The supernatant was discarded, and cell pellets were resuspended in 300 μL of TGY media. Then, 50 μL of E. coli donor or control culture and 50 μL of D. radiodurans recipient culture were directly mixed and spread on a conjugation plate, previously dried for 1 h, consisting of TGY media supplemented with diaminopimelic acid 60 μg mL ^-1 . After conjugation plates were incubated at 30°C for 3 h, the cells were scraped off with 1 mL of TGY media and adjusted to a final volume of 1 mL in a microfuge tube. Cell suspensions were serially diluted in TGY media and 100 μL was plated on both selective (TGY supplemented with chloramphenicol 5 μg mL ^-1 ) and nonselective (TGY) plates to select transconjugants and determine the conjugation frequency. Nonselective and selective plates were incubated at 30°C for 2 days and 3 days, respectively, and colonies were counted manually. Transconjugant Plasmid Isolation. D. radiodurans transconjugant colonies (10 from each experiment, 30 total) were passed twice on TGY plates supplemented with chloramphenicol (5 μg/mL), then inoculated into 5 mL of the same media and grown overnight. Alkaline lysis was performed using 3 mL of saturated culture as described [37], and the DNA was electroporated into E. coli Epi300 cells. The plasmids were induced to high copy number in E. coli in 5 mL of LB media supplemented with chloramphenicol (15 μg/mL) and arabinose (100 μg/mL) for 8 hours before isolating for analysis using the BioBasic EZ10 Spin Column Miniprep Kit. Transconjugant Plasmid Analysis. Plasmids were analyzed by multiplex PCR using the primers listed in Supplementary Table 4 using 10 μL of Qiagen MPX, 3 μL of primer mix, 6 μL of water and 1 μL of template diluted to 1 ng μL ^-1 . Thermocycler conditions were as follows: 95°C 5 min, 35 cycles of :94°C 30 sec, 60°C 90 sec, and 72°C 10 sec, then 72°C 10 min. Gel electrophoresis was performed by loading 2 uL of the multiplex on a 2% agarose gel at 90 kV for 60 min and was stained with ethidium bromide for visualization. Transconjugant plasmids were further analyzed with a diagnostic restriction digest using 0.2 μL of EcoRI-HF, 5 μL of ~100 ng/μL plasmid DNA, 2 μL Cutsmart buffer and 12.8 μL of water incubated at 37°C for 2 hours. Gel electrophoresis was performed by loading 10 uL of the digest on a 1% agarose gel at 100 kV for 120 min. Two transconjugant plasmids were analyzed by next-generation sequencing (MGH CCIB DNA Core, Massachusetts, USA) and compared to the sequence of the original pDEINO1 plasmid using the Benchling alignment tool (Benchling [Biology Software]. (2021). Retrieved from https://benchling.com). Spot Plating D. radiodurans. D. radiodurans was grown overnight in 5 mL cultures of TGY media supplemented with the appropriate antibiotics for plasmid maintenance. The cultures were diluted to an OD ₆₀₀ of 0.1 before performing 10-fold serial dilutions in TGY media up to 10- ⁵ dilution. Then, 5 uL of each dilution was plated on TGY plates supplemented with appropriate antibiotics and incubated at 30 degrees for 2-3 days. Plasmid Loss Stability Assay of D. radiodurans Transconjugants. One D. radiodurans transconjugant harbouring pDEINO1 was plated on TGY supplemented with chloramphenicol (5 μg mL ^-1 ) to obtain single colonies. A single colony was inoculated in 50 mL of the same media and grown with shaking to an OD ₆₀₀ of 0.5 at 30°C. The culture was diluted to an OD ₆₀₀ of 0.1 and 100 uL of a dilution series of 10 ^-1 to 10 ^-5 was plated on non-selective (TGY) and selective (TGY supplemented with chloramphenicol 5 μg mL ^-1 ) media. These plates were incubated at 30°C for 3 days and colonies were counted manually. From the diluted culture (OD ₆₀₀ of 0.1), 50 uL was used to subculture into 50 mL of fresh non-selective media and grown with shaking at 30°C for an additional ~13 generations to an OD ₆₀₀ of 0.5. This process was repeated for a total of ~40 generations. The ratio of D. radiodurans colonies on selective plates to colonies on non-selective plates after each subculturing event was reported as an indicator of plasmid loss. Additionally, 100 colonies from the selective and nonselective plates were struck onto TGY plates supplemented with chloramphenicol each day and the number of colonies unable to grow on selection was used as a second strategy to determine plasmid loss. Cloning the D. radiodurans MP1 Megaplasmid. Conjugation from E. coli to D. radiodurans was performed as described above using E. coli ECGE101 pTA-Mob pDEINO2 as the donor and D. radiodurans as the recipient, with D. radiodurans grown to an OD ₆₀₀ of 2 rather than 0.6. D. radiodurans transconjugants were selected by plating 100 μL onto 10 TGY plates supplemented with chloramphenicol (5 μg/mL). Three transconjugants were isolated and analyzed as previously described, transformed into E. coli Epi300 and, following induction in E. coli, plasmids were isolated using alkaline lysis. The cloned MP1 plasmids were analyzed by two multiplex PCRs, one amplifying fragments within the conjugative suicide plasmid, and one amplifying fragments within the MP1 megaplasmid. The plasmids were further analyzed by two diagnostic restriction digests using MfeI-HF and NheI-HF. Finally, total DNA from one clone was isolated using the Monarch Kit for HMW DNA Extraction from Bacteria (NEB#T3060) and confirmed by Nanopore sequencing. Gene Deletions. Conjugation from E. coli to D. radiodurans was performed as described above using E. coli ECGE101 pTA-Mob harboring pDEINO29 (ORF14075P), pDEINO30 (ORF15360P), pDEINO32 (MrrP) or pDEINO33 (ORF2230P) as the donor and D. radiodurans as the recipient. D. radiodurans transconjugants were selected on TGY supplemented with neomycin (5 μg/mL) for pDEINO29 and pDEINO33 or tetracycline (0.5 μg/mL) for pDEINO30 and pDEINO32. Transconjugants were isolated and analyzed by multiplex PCR with primers listed in Supplementary Table 4 using 10 μL of Qiagen MPX, 2 μL of primer mix, 6 μL of water, 1 μL of DMSO and 1 μL of alkaline lysis DNA. Thermocycler conditions were as follows: 95°C 5 min, 30 cycles of 94°C 30 sec, 60°C 90 sec, and 72°C 90 sec, then 72°C 10 min. Gel electrophoresis was performed by loading 2 uL of the multiplex on a 2% agarose gel at 110 kV for 50 min. D. radiodurans R1 Genomic DNA Isolation. D. radiodurans genomic DNA was isolated in agar plugs using the Bio-Rad CHEF Genomic DNA Plug Kit (Bio-Rad, CAT#170-3592) with an adapted protocol [37]. To prepare the plugs, 50 mL of D. radiodurans culture was grown to OD600 of 1.0, chloramphenicol (100 μg mL-1) was added and the culture was grown for an additional hour. The culture was centrifuged at 5000 × g for 5 min at 4°C. Cells were washed once with 1 M sorbitol in 1.5 mL Eppendorf tubes and centrifuged at 4000 RPM for 3 min. The supernatant was removed and cells were resuspended in 600 uL of protoplasting solution (for 10 mL: 4.56 mL of SPEM solution, 1000 μl Zymolyase-20 T solution (50 mg mL ^-1 dissolved in H ₂ O), 400 μL lysozyme (25 mg mL ^-1 ), 400 μl hemicellulase (25 mg mL ^-1 ), 50 μl β-Mercaptoethanol). The cell suspension was incubated for 5 min at 37°C and mixed with an equal volume of 2.0% low melting-point agarose in 1 × TAE buffer (40 mM Tris, 20 mM acetic acid and 1 mM EDTA) which was equilibrated at 50°C. Aliquots of 95 μl were transferred into plug molds (Bio-Rad, CAT#170– 3713) and allowed to solidify for 10 min at 4°C. Next, plugs were removed from the moldsinto 50 ml conical tube containing 5 mL of protoplasting solution and incubated for 45 min at 37°C. Next, plugs were washed with 25 ml of wash buffer (20 mM Tris, 50 mM EDTA, pH 8.0), and then incubated in 5 ml in Proteinase K buffer (100 mM EDTA (pH 8.0), 0.2% sodium deoxycholate, and 1% sodium lauryl sarcosine, 1 mg ml-1 Proteinase K) for 24 hr at 50°C. The plugs were washed 4 times with 25 mL of wash buffer for 30 min each at room temperature and incubated in wash buffer overnight at 4°C. The next day, the plugs were washed 4 times with 10X diluted wash buffer for 30 minutes each, then stored at 4°C in 10X diluted wash buffer. To isolate DNA from the plugs, two plugs were transferred to a 1.5 mL Eppendorf tube and were washed once with 10X diluted wash buffer for 1 hour, and once with TE buffer for 1 hour. The TE buffer was removed and the tube was incubated in a 42°C water bath for 10 min, followed by a 65°C water bath for 10 min. The tube was returned to the 42°C water bath for 10 min then 50 μL of TE buffer and 3 μL of β- agarase was added and flicked gently to mix. The plugs were incubated at 42°C for an hour. Another 50 μL of TE buffer was added to the tube and left for 2 hours at 42°C. The DNA was checked for purity by gel electrophoresis of 1 μL on a 1% gel, run at 100 kV for 40 min. DNA Sequencing and Analysis. D. radiodurans R1. The library was prepared using the SQK-LSK109 kit according to the manufacturer's protocol. An R9.4.1 flow cell was used for sequencing. Basecalling was performed using Guppy v4.2.2 (Oxford Nanopore Technologies) in high-accuracy mode. Genome assembly was performed with Flye v2.8.1 (Kolmogorow, 2019). The assembly was polished with one round of Racon (Vaser 2017) and one round of Medaka (Oxford Nanopore Technologies). E. coli pDEINO2-MP1. The library was prepared using the SQK-LSK109 kit and the EXP-NBD104 native barcoding kit according to the manufacturer's protocol. An R9.4.1 flow cell was used for sequencing. Basecalling was performed using Guppy v5.0.7 (Oxford Nanopore Technologies) in high-accuracy mode. Genome assembly was performed with Flye v2.8.1 in –meta mode[38]. The assembly was polished with one round of Racon (Vaser 2017) and one round of Medaka (Oxford Nanopore Technologies). Plasmids were extracted from the final assemblies, and aligned using minimap2[39] against the expected sequences to identify any mutations. Results A conjugation protocol has been developed as a direct method of DNA transfer from a recipient cell to D. radiodurans (Fig.1). To achieve this we first constructed a replicating plasmid, pDEINO1, by assembling a codon-optimized chloramphenicol acetyltransferase gene (cat) and an origin of replication [16] for D. radiodurans into the pAGE3.0 [31] multi-host shuttle (MHS) plasmid (Fig. 2A). A simple conjugation protocol was developed using an E. coli ΔdapA donor strain [31] to allow easy counter selection of the donor bacteria. The donor strain harbors the helper plasmid pTA-Mob [30] and the ~22 kb mobilizable plasmid, pDEINO1. Conjugation frequency was determined by comparing the number of D. radiodurans colonies that formed on media supplemented with chloramphenicol to the number of colonies on nonselective media. The mean conjugation frequency from E. coli to D. radiodurans was 1.12 x 10 ^-5 transconjugants per recipient (Fig. 2B). When E. coli harboring pDEINO1, but lacking pTA-Mob, was used as a donor for conjugation there were no transconjugant colonies on selective plates. There were also no transconjugant colonies formed when the recipient was plated with no E. coli donor. To confirm that the D. radiodurans transconjugants harbored pDEINO1, we isolated plasmids from 30 individual D. radiodurans transconjugant colonies. The DNA was transformed into E. coli Epi300, colonies were selected on media supplemented with chloramphenicol, and plasmids from 30 single colonies were induced to high copy number with arabinose. The plasmids were analyzed by multiplex PCR (Fig. 2C) and diagnostic restriction digest (Fig. 2D), both of which showed the expected banding patterns following gel electrophoresis. Additionally, sequencing analysis of plasmids from two transconjugant colonies showed no mutations when compared to the original pDEINO1 sequence. These results demonstrate that replicating plasmids can be successfully conjugated from E. coli to D. radiodurans with no gross rearrangements or point mutations. Furthermore, the codon-optimized chloramphenicol acetyltransferase gene is an effective plasmid-based marker for selection of D. radiodurans transconjugants. This strategy of using conjugation to deliver replicating plasmids circumvents the difficulties associated with transforming DNA into strains containing active restriction systems. To determine the sensitivity of D. radiodurans to chloramphenicol, five transconjugant colonies were spot plated alongside the wild-type strain on varying concentrations of chloramphenicol (Fig. 2E). We found that chloramphenicol at a concentration of 5 μg mL ^-1 inhibited the growth of wild-type D. radiodurans, while having only a minor negative effect on transconjugants; however, any additional increase in chloramphenicol concentration significantly impacted transconjugant growth. To demonstrate that the strains could be cured of the plasmid, we performed a plasmid-loss stability assay on one transconjugant colony grown without selection in liquid media for approximately 40 generations. At 13-generation intervals, the culture was plated on media with and without chloramphenicol and 100 colonies from each treatment were restruck onto selective plates. We showed that when growing without selection, on average 44% of cells lose the pDEINO1 plasmids every 13 generations (Table 1). Plasmid loss is beneficial when employing an engineering strategy such as those that depend on the recycling of plasmids or that require plasmids temporarily for expression of an endonuclease or Cas9, for instance. Since our plasmids could be maintained when propagating with the antibiotic marker, the development of auxotrophic strains and compatible complementation plasmids could be beneficial to develop in the future. Although horizontal gene transfer to D. radiodurans has been predicted from comparative genomics studies [40], suggesting that conjugation to this organism has occurred naturally before, our results are the first demonstration harnessing conjugative machinery for delivery of replicating and nonreplicating plasmids in a laboratory setting. In order to design plasmid-based systems utilizing this new conjugative delivery method and to increase its potential for engineering D. radiodurans, we tested four additional antibiotic markers. Previous studies have demonstrated the use of nptII (Kan/Nm ^R ), aadA (Spec ^R ), cat (Cm ^R ) and aac(3) (Gen ^R ) to select for genomic integration of transgenes[41]. Therefore, we cloned nptII, tetR/A, aadA1, or aacC1 (conferring resistance to kanamycin/neomycin, tetracycline, streptomycin, and gentamicin, respectively) downstream of the codon-optimized chloramphenicol marker onto separate replicating plasmids (Fig.3A). These plasmids, called pDEINO1, pDEINO3, pDEINO4 and pDEINO5, were conjugated to D. radiodurans from E. coli and transconjugant colonies were selected on plates supplemented with chloramphenicol. Individual colonies harboring each of the different plasmids were grown in liquid media alongside wild type D. radiodurans, and 10-fold serial dilutions were spotted onto plates supplemented with the aforementioned antibiotics. As shown in Fig. 3B, all strains harboring shuttle vectors conferred effective resistance to chloramphenicol and their respective antibiotic, while exhibiting sensitivity to the other three antibiotic supplements. Since nptII confers resistance to two antibiotics, the strain carrying pDEINO1 was also tested on media supplemented with kanamycin, though neomycin caused slightly less growth inhibition to transconjugants (Fig.6). We observed that supplementing gentamicin at a concentration as little as 2 μg mL ^-1 sufficiently inhibited wild type D. radiodurans for our applications, although there was some breakthrough at the lowest dilution on the spot plates. Furthermore, changing the concentration by only 1 μg mL ^-1 significantly impacted the growth of D. radiodurans harboring pDEINO5. Increasing gentamicin concentration to 3 μg mL- ¹ severely inhibited growth, while decreasing the concentration to 1 μg mL ^-1 did not inhibit wild type D. radiodurans when compared to strains harboring the plasmid (data not shown). Gene deletions and editing have previously been demonstrated in D. radiodurans The first chromosomal mutants in D. radiodurans were made by integrating E. coli plasmids into the D. radiodurans genome by cloning host DNA sequences into the plasmids [17]. However, duplicated insertions were often heterozygous and can be deleted by intrachromosomal recombination events [25]. Targeted integration to create mutants became possible after the genome was sequenced [42]. Expression vectors have also been integrated into D. radiodurans to engineer it for pollutant degradation or detoxification of mercury and toluene [8,9]. More recently, multiple knockouts of genes replaced by selection markers have been demonstrated by recombination of nonreplicating plasmid in D. radiodurans [43], as well as a Cre-lox recombination system [22]. With an efficient plasmid delivery method and a collection of selection markers to work with, we developed a conjugative plasmid-based protocol for generating gene deletions in D. radiodurans. We created a nonreplicating plasmid to include a construct of interest, in this case nptII or tetR/A resistance genes, between two regions of homology from the D. radiodurans genome flanking a gene targeted for deletion. In the interest of improving transformation efficiency, four restriction endonuclease genes in the D. radiodurans genome, which have been identified and individually deleted in previous studies [44–46], were chosen as deletion targets. These genes include Mrr and ORF2230 located on chromosome 1, ORF14075 on chromosome 2, and ORF15360 on the MP1 megaplasmid (Fig.4A). Prior to plasmid construction we resequenced our D. radiodurans R1 strain to ensure accurate homology sequences were incorporated into the plasmid designs. Genomic DNA was isolated using agar plug DNA isolation and was sequenced using Oxford Nanopore MinION sequencing technologies. Interestingly, we found that our plasmids did not contain the large insertions as reported in the PacBio sequencing results [13], and the plasmid sizes more closely resembled the original sequence published by White et al. [12]. All four nonreplicating plasmids were constructed with a backbone for replication and selection in E. coli and Saccharomyces cerevisiae, an oriT for conjugative transfer, and a deletion cassette containing a selectable marker – neomycin or tetracycline – between 1 kb regions of homology flanking the gene of interest (Figure 4B). Following conjugative delivery, transconjugants were selected with the respective antibiotic, DNA was isolated from D. radiodurans and screened for genomic gene deletions using multiplex PCR. Three amplicons could be observed in the multiplex, which reside within the following elements: selectable marker, plasmid backbone, and wild type restriction endonuclease gene. Two transconjugants were screened for each single deletion event and PCR showed amplification of only the selectable marker, indicating successful integration of the selectable marker, deletion of the targeted gene, and loss of the plasmid backbone (Fig.3C). To show that this system could also be used to generate multiple deletions in the same strain, we performed conjugation using an ORF15360 transconjugant as the recipient and a donor E. coli harboring pDEINO29 targeting ORF14075. Transconjugants were selected on TGY media supplemented with both neomycin and tetracycline and their DNA was screen using the same multiplex PCRs as were used for the single deletion, confirming that both ORF15360 and ORF14075 genes were replaced with the tetracycline and neomycin markers respectively, and the plasmid backbone was lost (Fig.4C). Successful deletion of multiple genes demonstrated that this technique could be implemented to sequentially delete genes involved in recognizing and digesting foreign or synthetic DNA in the cell in order to develop a restriction-free D. radiodurans strain. To engineer D. radiodurans as an industrial platform organism, streamlined strains, such as those developed for Mycoplasma mycoides JCVI-syn3.0, are developed by building synthetic genomes. Creating a minimal D. radiodurans cell will likely require the use of intermediate host organisms, such as E. coli, yeast, or S. meliloti to hold the DNA and carry out efficient genetic modification and cloning [31,37,47]. To this end, we aimed to demonstrate that large DNA fragments from the D. radiodurans genome could be cloned in E. coli. We built another nonreplicating plasmid called pDEINO2, and used our conjugation-based genome integration strategy to clone the native MP1 megaplasmid, 178 kb in length with a notable G+C content of ~62%, from D. radiodurans. To facilitate recombination and integration of the entire plasmid, pDEINO2 contained a 1 kb region of homology to the middle of the McrC gene on the MP1 megaplasmid (Fig.5A). Following assembly, pDEINO2 was transformed into the ΔdapA E. coli donor strain and delivered to D. radiodurans via conjugation (Fig. 5B). We confirmed recombination of pDEINO2 at the McrC gene by extracting DNA from transconjugants selected on TGY plates supplemented with chloramphenicol and performing a multiplex PCR (Fig. 5C, Fig.7). Plasmid DNA was then transformed into E. coli, induced to high copy number and colonies were screened to verify the MP1 megaplasmid had been transferred and propagated without compromising genomic integrity. The pDEINO2 plasmid was confirmed to be integrated at the correct location using two diagnostic digests (Fig.5D) and multiplex PCR that amplify regions of pDEINO2 and the D. radiodurans MP1 megaplasmid (Fig.5E). In particular, the presence of the 7733 bp band in the MfeI-HF digest was a strong indicator of plasmid integration at the McrC locus, as there is an MfeI site just outside the integration site and in the pDEINO2 backbone that creates a unique band size that would otherwise not be present in the wild type MP1 plasmid. The same is true for the 8112 bp band in the NheI-HF digest. Finally, we extracted total DNA from one E. coli clone harboring the pDEINO2-cloned MP1 plasmid and sequenced it using the Oxford Nanopore MinION. The cloned MP1 plasmid was assembled and sequence data confirmed that pDEINO2 successfully integrated at the intended target site and the final plasmid size was ~190 kb. Coverage for the plasmid was found to have a mean of 1098X, while sequencing depth for the genome had a mean of 152X, indicating ~7X multiplicity of the plasmid in E. coli following induction. These results indicated that large regions of the D. radiodurans genome, including entire replicons, can be effectively cloned and propagated in E. coli. In summary, we have demonstrated conjugation from an E. coli donor host as a method of DNA delivery to D. radiodurans and created several conjugation-based tools for genome engineering. The tools described in this disclosure will rapidly advance the development of designer D. radiodurans strains for synthetic biology applications, as well as facilitate foundational biology studies that identify and characterize the features that make this species uniquely indestructible. EXAMPLE 2 Overview Many microorganisms have restriction-modification (R-M) systems as part of the bacterial immune system, protecting against foreign DNA molecules[48]. Putative R-M systems in D. radiodurans R1 have been identified throughout its two chromosomes and two plasmids, which has been summarized on REBase (http://rebase.neb.com/, reference #22767)[49]. The genome contains four predicted Type II and two Type IV R-M systems containing restriction endonucleases, as well as a lone methyltransferase on the CP1 plasmid. Some of the R-M systems have been further characterized empirically[44–46] and previous studies have shown that these R- M systems may be preventing the efficient transformation of D. radiodurans [50]. In one embodiment, the present disclosure provides for a Seamless Loss of Integrated Cassettes using Endonuclease Cleavage and Recombination (SLICER) method that allows for the recycling of selection markers and demonstrated its use by sequential deletion of four of the six R- M systems. Following the transformation of a neomycin selectable marker to replace the fifth R- M system, a final D. radiodurans strain lacking five of the six R-M systems was created. Deletion of these systems did not result in growth inhibition of these D. radiodurans strains, and improved transformation efficiency of the pRAD1 plasmid (6 kb). Use of the SLICER method for engineering D. radiodurans enables the deletion of any essential or nonessential gene of interest (GOI) and ultimately lead to further strain improvements. Methods Microbial Strains and Growth Conditions. Deinococcus radiodurans R1 was grown at 30°C in TGY medium (5 g L ^–1 tryptone, 3 g L ^–1 yeast extract, 1 g L ^–1 potassium phosphate dibasic, and 2.5 mL of 40% w/v glucose) supplemented with appropriate antibiotics (chloramphenicol, 5 μg mL ^–1 ; neomycin, 5 μg mL ^–1 ). Escherichia coli (Epi300, Lucigen) was grown at 37°C in Luria Broth (LB) supplemented with chloramphenicol, 15 μg mL ^–1 . Escherichia coli ECGE101 (ΔdapA)[31] was grown at 37°C in LB media supplemented with DAP, 60 μg mL ^–1 , and appropriate antibiotics (chloramphenicol, 15 μg mL ^–1 ; gentamicin, 40 μg mL ^–1 ). Saccharomyces cerevisiae VL6-48 (ATCC MYA-3666: MATα his3-Δ200 trp1-Δ1 ura3–52 lys2ade2–1 met14 cir ⁰ ) was grown at 30°C in 2X YPAD rich medium (20 g L ^–1 yeast extract, 40 g L ^–1 peptone, 40 g L ^–1 glucose, and 80 mg L ^–1 adenine hemisulfate), or in complete minimal medium lacking histidine supplemented with 60 mg L ^–1 adenine sulfate (Teknova Inc.) with 1 M sorbitol. All strains created in this study are summarized in Table 5. Deinococcus radiodurans R1 was grown at 30°C in TGY medium (5 g L ^-1 tryptone, 3 g L- ¹ yeast extract, 1 g L ^-1 potassium phosphate dibasic, and 2.5 mL 40% w/v glucose) supplemented with appropriate antibiotics (chloramphenicol 5 μg mL ^-1 , neomycin 5 μg mL ^-1 , tetracycline 0.5 μg mL ^-1 ). Escherichia coli (Epi300, Lucigen) was grown at 37°C in Luria Broth (LB) supplemented with chloramphenicol 15 μg mL ^-1 . Escherichia coli ECGE101 (ΔdapA) [31] was grown at 37°C in LB media supplemented with diaminopimelic acid (DAP) 60 μg mL ^-1 and appropriate antibiotics (chloramphenicol 15 μg mL ^-1 and/or gentamicin 40 μg mL ^-1 ). Saccharomyces cerevisiae VL6−48 (ATCC MYA-3666: MATα his3-Δ200 trp1-Δ1 ura3−52 lys2 ade2−1 met14 cir ⁰ ) was grown at 30°C in 2X YPAD rich medium (20 g L ^-1 yeast extract, 40 g L ^-1 peptone, 40 g L ^-1 glucose, and 80 mg L ^-1 adenine hemisulfate), or in complete minimal medium lacking histidine supplemented with 60 mg L ^-1 adenine sulfate (Teknova Inc.) with 1 M sorbitol. CaCl ₂ Transformation of D. radiodurans. For competent cells: A 50 mL culture of D. radiodurans was grown to an OD ₆₀₀ of 0.2. The culture was transferred to a 50 mL falcon tube and centrifuged at 3000 g for 15 min at 4°C. The supernatant was discarded, and the pellet was resuspended in 250 μL of ice-cold 0.1 M CaCl ₂ 15% glycerol solution using gentle agitation. The competent cells were aliquoted in 50 μL increments, frozen in a -80°C ethanol bath and stored at -80°C. For transformation: 50 μL of competent cells per reaction were thawed on ice for 15 min. Then, 5 μL of transforming DNA (linear PCR product or plasmid) was mixed with the competent cells. The mixture was incubated on ice for 30 min then heat shocked in a 42°C water bath for 45 seconds. The tubes were returned to ice for 1 min and 1 mL of 2X TGY media was added to each tube. The recovery cultures were transferred to a 50 mL falcon tube and grown with shaking at 30°C for 2 hours at 225 rpm. Finally, 300 μL of the transformation mixture was plated on TGY media with appropriate supplements (chloramphenicol 3 μg mL ^-1 or neomycin 5 μg mL ^-1 and/or X-Gal 40 μg mL ^-1 ) and incubated at 30°C for 2-3 days. Colonies were counted manually. Conjugation from E. coli to D. radiodurans. Conjugation from E. coli to D. radiodurans was performed as previously described [51], with the following modifications. The donor strain was E. coli ECGE101 ΔdapA[31] harbouring pTA-Mob [30] and pSLICER. The D. radiodurans R1 recipient strains with the integrated SD cassettes were grown in TGY media supplemented with neomycin (5 μg mL ⁻¹ ) prior to conjugation. The selective plates were TGY media supplemented with chloramphenicol (3 μg mL ⁻¹ ). Plasmid Design and Construction. All plasmids in this study (Table 3) were constructed from PCR amplified DNA fragments assembled using a yeast spheroplast transformation method as previously described (Cochrane, R. R. et al. Rapid method for generating designer algal mitochondrial genomes. Algal Res.50, 102014 (2020). The primers used to amplify the fragments for plasmid assembly (Table 4) contained 20 bp binding and 40 bp of overlapping homology to the adjacent DNA fragment. Following assembly, DNA was isolated from S. cerevisiae and the plasmid pool was electroporated into E. coli Epi300. Plasmids from individual colonies were screened for correct assembly using multiplex PCR and diagnostic restriction digest. All plasmids were built to contain a pCC1BAC-yeast backbone allowing replication and selection in E. coli (chloramphenicol) and S. cerevisiae (-HIS) with a low-copy E. coli origin that can be induced to high copy with arabinose. They also have an origin of transfer (oriT) necessary for conjugation. pSD1-4: nonreplicating plasmids containing two ~1 kb regions of homology flanking ORF14075, Mrr, ORF15360, and Mrr2, respectively, amplified from wild-type D. radiodurans genomic DNA. Between the homology regions on the plasmids is an I-SceI recognition site, a selective marker (nptII) and visual screening marker (lacZ) amplified from pDEINO1 and pET-24α(+)-lacZ, respectively, and an 80 bp duplication of the 3’ end of homology region 1. The aforementioned elements make up the SD cassette. These plasmids also contain a second selective marker for D. radiodurans outside of the SD cassette, tetR/A or aadA1 amplified from pDEINO3 and pDEINO4, respectively (Brumwell, S. L., Van Belois, K. D., Giguere, D. J., Edgell, D. R. & Karas, B. J. Conjugation-Based Genome Engineering in Deinococcus radiodurans. ACS Synth. Biol. 11, (2022)). pSLICER: replicating plasmid built to contain a D. radiodurans codon-optimized cat gene under the control of a constitutive promoter (drKatA) and origin of replication amplified from pDEINO1 (Brumwell, S. L., Van Belois, K. D., Giguere, D. J., Edgell, D. R. & Karas, B. J. Conjugation-Based Genome Engineering in Deinococcus radiodurans. ACS Synth. Biol. 11, (2022)). A synthesized D. radiodurans codon-optimized I-SceI endonuclease gene was also incorporated on this plasmid under the control of the PDR_2508 promoter and terminator set (Chen, A. et al. Discovery and characterization of native Deinococcus radiodurans promoters for tunable gene expression. Appl. Environ. Microbiol.85, (2019)). D. radiodurans Genomic DNA Isolation. Alkaline lysis was performed using 3 mL of saturated culture as previously described[52] to extract D. radiodurans genomic DNA for analysis. Multiplex PCR Analysis of D. radiodurans Knockouts. Multiplex PCR analysis was performed according to the manufacturer’s instructions for “Standard Multiplex PCR” (Qiagen Multiplex PCR Handbook) with the following modifications and the primers listed in Table 4. A final volume of 20 μL was used and reaction mix components were adjusted accordingly. A volume of 1 μL of undiluted template DNA and 1 μL of dimethyl sulfoxide (DMSO) was used in the reaction mix. Thermocycler conditions were chosen according to the “Universal Multiplex Cycling Protocol” with the initial activation step decreased to 5 min, using an annealing temperature of 60°C, and 30 cycles. Gel electrophoresis was used to visualized 2 μL of the PCR product on a 2% agarose gel. Spot Plating D. radiodurans. D. radiodurans was grown overnight in 5 mL cultures of TGY media supplemented with the appropriate antibiotics (none, neomycin or chloramphenicol). The cultures were diluted to an OD ₆₀₀ of 0.1 before performing 10-fold serial dilutions in TGY media up to 10 ^-5 dilution. Then, 5 μL of each dilution was plated on nonselective TGY media and/or TGY media supplemented with appropriate antibiotics and incubated at 30°C for 2-3 days. D. radiodurans Growth Curve and Doubling Time Calculation. Growth rates were evaluated for D. radiodurans strains: wild type, ΔRM1, ΔRM1-2, ΔRM1-3, ΔRM1-4, and ΔRM1- 5 Nm ^R . Single colonies were inoculated into 5 mL of liquid TGY media and grown overnight at 30°C with shaking at 225 rpm. Cultures were diluted to an OD ₆₀₀ of 0.1 in the same media, and 200 μL of each culture was aliquoted into a 96-well plate, along with a TGY media only control. In the Epoch 2 (BioTek, USA) plate reader, strains were grown at 30°C with continuous, orbital shaking (559 cpm). Absorbance (A ₆₀₀ ) measurements were taken every 15 min for 24 h for a total of 97 readings using Gen5 data analysis software version 3.08.01 (Biotek, USA). This experiment was performed with three biological replicates, each with two technical replicates. Growth curves were plotted with data points representing the average of six measurements for each strain with error bars representing standard error of the mean. For simplicity, every other time point was omitted; therefore, readings are presented for every 30 min and the curve is cut off at the 17-hour time point when cultures approached end point density. The doubling time of each replicate was determined using the R package Growthcurver (Sprouffske, K. & Wagner, A. Growthcurver: An R package for obtaining interpretable metrics from microbial growth curves. BMC Bioinformatics 17, (2016)). The doubling time is reported as an average of the six replicates for each strain, and the standard deviation was calculated. RESULTS We sought to develop a strategy for generating seamless gene deletions in D. radiodurans. To achieve this, we developed a method called SLICER. In one embodiment of SLICER, a nonreplicating multi-host shuttle plasmid, termed the Seamless Deletion plasmid (pSD), is first built specifically for the targeted DNA region or GOI (Fig.8B). The key component of this plasmid will herein be referred to as the Seamless Deletion (SD) cassette, which contains a neomycin resistance gene and lacZ (β-galactosidase) marker for selection and visual screening in D. radiodurans flanked by two regions homologous to the D. radiodurans genome. These about 1 kb homology regions are the sequences upstream and downstream of the target sequence. These two homology regions are referred to as first homology region and second homology region. Following the first homology region, there is an 18-bp I-SceI endonuclease recognition site and prior to the second homology region there is a duplication of the last 80 bp of the first homology region. A second selective marker for D. radiodurans is located on the plasmid backbone outside of the SD cassette. The first step in the SLICER method (Fig.8A) is the integration of the SD cassette into the D. radiodurans genome at the target locus (i.e., at the GOI). The SD cassette, a linear DNA molecule, can be PCR amplified and delivered via chemical transformation into D. radiodurans. Alternatively, the whole pSD plasmid could be delivered via conjugation. Next homologous recombination of the two homology regions (i.e., the first and the second homology regions) with the corresponding genomic regions of D. radiodurans results in integration of the SD cassette into the D. radiodurans genome, replacing the target GOI. D. radiodurans transformants containing the SD cassette can be selected on TGY media supplemented with neomycin (5 µg mL ^-1 ) and X- Gal (40 µg mL ^-1 ) and appear blue in colour due to the expression of the lacZ gene. The resulting strain is referred to as D. radiodurans + SD. The second step in the SLICER method is the removal of the SD cassette facilitated by the introduction of a pSLICER plasmid (Fig. 8C). We constructed the replicating helper plasmid, pSLICER, containing a codon-optimized I-SceI endonuclease, an origin of replication and a chloramphenicol selective marker for D. radiodurans (Fig.8A). I-SceI was chosen because there are no recognition sites present in the wild-type genome of D. radiodurans. The I-SceI endonuclease was designed under the regulation of the PDR_2508 promoter and terminator set as it was shown to have high expression in D. radiodurans but low expression in E. coli[21]. This plasmid is then transformed into an E. coli ΔdapA strain harbouring the conjugative plasmid pTA- Mob[30]. Conjugation of the pSLICER plasmid from the E. coli donor strain to D. radiodurans + SD is then performed. Expression of the I-SceI endonuclease leads to cleavage at the I-SceI recognition sequence within the SD cassette which stimulates homologous recombination between the first homology region and the 80-bp duplicated region of the first homology region. Transconjugants can be selected on TGY media supplemented with chloramphenicol (3 µg mL ^-1 ) and X-Gal. Contrary to the previous screening, transconjugants that have had the SD cassette removed via homologous recombination should appear pink since they have lost the lacZ gene. The resulting strain is referred to as D. radiodurans + SLICER. The codon-optimized I-SceI endonuclease was proven to be functional in D. radiodurans and essential for SD cassette excision and ultimately for the success of the SLICER method. To determine the frequency of SD cassette loss from the D. radiodurans genome following integration, dilutions of D. radiodurans ΔRM1-4 Nm ^R , harbouring the SD cassette, were plated on nonselective and selective media (Table 6). Due to the presence of lacZ in the SD cassette, blue colonies should be indicative of those carrying the SD cassette while pink colonies indicate loss of the cassette. The percentage of D. radiodurans colonies that appeared pink with and without antibiotic selection were 1.1% and 2.1%, respectively, indicating the occurrence of natural SD cassette loss or mutation following propagation. The pink colonies obtained from both nonselective and selective plates were further analyzed by streaking them onto selective media (data not shown). All colonies were able to grow on selective media, indicating that while these colonies appeared to have lost or mutated the lacZ gene, the neomycin marker in the SD cassette was still functional. As such, the integrated SD cassettes appear to be quite stable and spontaneous loss of these cassettes could not be easily obtained by growing cultures without selective pressure. As further confirmation that the I-SceI endonuclease is required for excision of the SD cassette in the SLICER method, conjugation of pSLICER and a control plasmid, pDEINO1, was performed to D. radiodurans ΔRM1-4 Nm ^R with the integrated SD cassette (Fig. 12). The pDEINO1 plasmid contains all of the same components as pSLICER including a D. radiodurans origin of replication and chloramphenicol marker but lacks the I-SceI endonuclease. When this plasmid was conjugated to D. radiodurans, all transconjugant colonies appeared blue, indicating that they still harbored the SD cassette. Conversely, when the pSLICER plasmid was conjugated to D. radiodurans, all transconjugant colonies appeared pink, indicating that the SD cassette had been lost. This allowed us to conclude that the codon-optimized I-SceI endonuclease is functional in D. radiodurans and is essential for SD cassette excision. The final step in the SLICER method is to cure the strain of the pSLICER plasmid. The D. radiodurans + SLICER strain is grown in nonselective media overnight and dilutions are subsequently spot plated on nonselective media. Resulting single colonies are then struck on nonselective media as well as TGY media supplemented with either chloramphenicol or neomycin. The colonies are confirmed to be cured of the plasmid when growth is observed on nonselective plates but not on selective plates. At the end of the seamless deletion process, the resulting D. radiodurans ΔGOI strain will have the target gene deleted with no remnants of the process remaining in the genome or the cell. The entire SLICER method can be completed in approximately 2 weeks and one embodiment of the step-by- step protocol is summarized in Fig.13. Using the seamless deletion strategy outlined in this disclosure, we performed the sequential deletion of four out of the six R-M system genes in the D. radiodurans genome (Figs. 9A to 9C), with the fifth R-M system (ORF2230) subsequently deleted using homologous- recombination based integration of a neomycin marker. Four nonreplicating SD plasmids, named pSD1-pSD4, were built for each R-M target gene: ORF14075, Mrr, ORF15360, and Mrr2, which will herein be called RM1, RM2, RM3 and RM4, respectively. These target genes were named numerically in the order that they were used to generate deletions. Each plasmid contains the same elements apart from the homology regions, which are specific to each target gene. It should be understood that all 6 R-M systems can be deleted by the methods of the present disclosure. Gene deletion analysis of all four knockout strains is shown in Fig.9C for the deletion of RM1, RM2, RM3 and RM4 resulting in the creation of D. radiodurans ΔRM1, ΔRM1-2, ΔRM1- 3 and ΔRM1-4 strains, respectively. Following Step 1, 2 and 3 of the SLICER protocol (as outlined in Fig. 8), the D. radiodurans genome was analyzed to confirm insertion of the SD cassette, removal of the SD cassette, and curing of the pSLICER plasmid. Analysis was conducted by spot plating dilutions on nonselective media, media supplemented with neomycin and media supplemented with chloramphenicol, all of which contained X-Gal (Fig. 9B). In addition, multiplex PCR analysis was performed on DNA extracted from one individual colony for each seamless deletion event (RM1-4) (Fig. 9C). If present in the examined DNA, the multiplex PCR should amplify a 150 bp amplicon at a non-target site in the D. radiodurans genome, a 300 bp amplicon within the neomycin marker on the SD cassette, a 500 bp amplicon within the target gene (RM1-4), and/or a 650 bp amplicon within the pSLICER backbone. The position and size of the expected amplicons following each step of the seamless deletion strategy are depicted in Fig.9A. For the RM1 multiplex, a wild-type genomic DNA control is used, and for all subsequent multiplex analyses the cured strain from the previous deletion was used as a control (e.g., D. radiodurans ΔRM1 DNA is used as a control for analysis of D. radiodurans ΔRM1-2). From the analyses of the RM1-RM4 deletions (Fig.9C), we observed that the wild-type D. radiodurans strain was only able to grow on nonselective media, appeared pink in colour, and the PCR results showed amplification of the genomic DNA control and target gene amplicons. Multiplex PCR performed on the pSD1-4 plasmids containing the SD cassette showed amplification of the neomycin marker and backbone amplicons. Following integration of the SD cassette, D. radiodurans + SD was able to grow on the nonselective and neomycin supplemented media, and appears blue in colour on both. The PCR results show amplification of the genomic control and notably, there is no amplification of the target gene amplicon. After conjugating in the pSLICER plasmid, D. radiodurans + SLICER is able to grow on nonselective and chloramphenicol supplemented media, but not neomycin supplemented media. With the loss of the SD cassette, the colonies once again appear pink. The PCR results show amplification of the genomic control and backbone amplicons. Notably, there is no amplification of the neomycin marker amplicon. Finally, curing of the pSLICER plasmid from D. radiodurans ΔRM only allows for growth on the nonselective plate as the strain no longer contains the SD cassette or the pSLICER plasmid, and colonies appear pink as a result. Multiplex PCR shows amplification only of the genomic control amplicon, indicating that the pSLICER plasmid was successfully cured. Following the fourth deletion, the fifth R-M system was deleted using homologous- recombination based integration of a neomycin marker using the cassette from pDEINO10 previously used to delete ORF2230[51]. The final D. radiodurans ΔRM1-5 Nm ^R strain can be propagated with neomycin selection (Fig.10A). Further confirmation that all four genes (RM1-4) have been seamlessly deleted in the D. radiodurans ΔRM1-4 strain was performed using multiplex PCR (Fig. 10B). The sixth R-M system that has not yet been deleted is mcrBC on the MP1 megaplasmid. Nevertheless, it should be understood that the method of the present disclosure can be used to delete any and/or all of the R-M systems of D. radiodurans. Physiological analysis of D. radiodurans ΔRM strains was performed by testing their growth in liquid TGY media. The growth phenotype of D. radiodurans ΔRM strains compared to wild type revealed no significant difference based on the growth curve, endpoint density or calculated growth rates (Fig.11A). This suggests that removal of the first five R-M system genes did not result in any growth deficits in D. radiodurans, which is promising if this strain (or the full restriction minus strain) are to be used as synthetic biology chassis in the future. Transformation of D. radiodurans ΔRM strains was performed using the ~6 kb pRAD1 plasmid to determine if these strains have improved transformation efficiency compared to wild type. Heat shock transformation was performed using 850 ng of plasmid DNA isolated from E. coli Epi300 into wild type and all five ΔRM strains (Fig.11B). These results indicate that through the deletion of five R-M systems, we were able to improve transformation by approximately 3- fold from wild type to D. radiodurans ΔRM1-5 Nm ^R with an average transformation efficiency of 3.86 x 10 ¹ and 1.27 x 10 ² CFU/μg DNA, respectively. In summary, we have created the first seamless gene deletion strategy for D. radiodurans and demonstrated that SLICER can be used for the sequential deletion of endogenous genes. Using this seamless deletion method, homozygous insertions and deletions can be made rapidly across all copies of the D. radiodurans genome, and it is the first report of the I-SceI endonuclease being used in these bacteria. We used the SLICER method to create a D. radiodurans strain with four restriction-system genes seamlessly deleted, and a fifth gene replaced with a selective marker. Physiological analysis of these strains showed no growth deficit and improved transformation efficiency. The SLICER method is invaluable for D. radiodurans engineering and allows for the seamless deletion of any DNA target of interest, including the remaining R-M systems. Table 1. Plasmid-loss stability assay of D. radiodurans harboring pDEINO1 over 40 generations. Table 2. List of strains Table 3. List of plasmids

Table 4. List of oligonucleotides. The bold, underlined sequence in the assembly primers represents the binding portion of the primer, while the remainder of the sequence is the hook or homology region to the adjacent fragment.

Table 5. Deinococcus radiodurans strains created in Example 2

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The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.