FR3136481A1 - METHODS AND COMPOSITIONS FOR DELIVERING NUCLEIC ACID - Google Patents

Abstract

The present invention relates to improved methods of delivering DNA to target bacteria and archaea. Fig. 1

Description

METHODS AND COMPOSITIONS FOR DELIVERING NUCLEIC ACID

The present invention relates to improved methods of delivering DNA to target bacteria and archaea.

Bacteria and archaea, such as those found in human and animal microbiota or environments, encode methyltransferase enzymes that protect host bacterial DNA from DNA cleavage by restriction modification (RM) systems encoded by the host. These RM systems target DNA that is not methylated in the same way as host DNA. RM systems enzymes cleave DNA at specific motifs within the DNA, and site-specific cleavage is prevented by methylation. In laboratory bacteria, RM systems can be modified to allow more efficient transformation of foreign DNA, but this is not possible for delivery to bacteria in natural environments such as the microbiome.

WO 2016/205276 discloses methods for methylating bacteriophage or phagemid DNA in production strains prior to delivery.

The invention concerns:

A carrier bacteria comprising a conjugative plasmid, the plasmid encoding a methyltransferase enzyme.

A method of delivering DNA from a carrier bacterium to a target bacterium by conjugation, the method comprising contacting the target bacterial species with the carrier bacterium as disclosed herein, wherein the target bacterium comprises a system of restriction modification that cleaves DNA that is unmethylated at a target site for methylation of the methyltransferase enzyme.

A bacteriophage encoding a methyltransferase enzyme, optionally wherein the methyltransferase enzyme protects against a restriction modification system found in a target bacteria that is pathogenic in humans or animals, and/or in a target bacteria that is associated with biofouling (in English: “biofouling”) or microbial corrosion.

A method of delivering bacteriophage DNA to a target bacteria, the method comprising contacting the target bacterial species with a bacteriophage encoding a methyltransferase enzyme, wherein the target bacteria comprises a restriction modification system that cleaves DNA that is unmethylated at a target site for methylation of the methyltransferase enzyme.

A process for methylating bacteriophage DNA or plasmid or phagemid DNA, the process comprising:

introducing into a production bacterium (i) a polynucleotide encoding a plurality of methyltransferases, or (ii) polynucleotides which together encode a plurality of methyltransferases,

the production, thereby, of a modified production bacterium expressing at least 2 introduced methyltransferases;

infecting the engineered production bacterium with a bacteriophage particle comprising bacteriophage DNA or phagemid DNA or plasmid DNA, thereby methylating said bacteriophage DNA or phagemid DNA or plasmid DNA; And

producing a bacteriophage particle comprising bacteriophage DNA or phagemid DNA or plasmid DNA having a modified methylation pattern, optionally purifying the bacteriophage particle comprising bacteriophage DNA bacteriophage or phagemid DNA or plasmid DNA.

A pharmaceutical composition comprising a/a carrier bacteria or bacteriophage or phagemid or plasmid as disclosed herein in combination with a pharmaceutically acceptable carrier or excipient.

A method of preventing or treating a disease caused by a pathogenic target bacteria in humans or animals, the method comprising the provision of a bacteriophage, a phagemid or a carrier bacteria or a plasmid or a pharmaceutical composition as described here to a human or animal in need thereof.

A/a bacteriophage, phagemid or carrier bacteria or plasmid or pharmaceutical composition as disclosed herein for use in a process for preventing or treating a disease caused by a pathogenic target bacteria in humans or animals, the process comprising: the provision of the bacteriophage, phagemid or the supporting bacteria or pharmaceutical composition to humans or animals in need.

A method of destroying a target bacterium or of targeted modification of the genome of a target bacterium, the method comprising providing a bacteriophage, phagemid or carrier bacteria or plasmid as disclosed herein to the target bacteria, in which the target bacteria is included in a naturally occurring population associated with microbial corrosion or biofouling of a substrate or fluid in an industrial or domestic system.

A method for targeted modification of a target bacterial species or strain, the method comprising bringing a target bacteria into contact with a/a support bacteria, bacteriophage or phagemid or plasmid as disclosed herein, so that the target sequence of the target bacterial species is modified, where the plasmid, phage or conjugable phagemid further comprises a CRISPR array modifying the target cell.

Other characteristics, details and advantages of the invention will appear on reading the detailed description below, and on analyzing the appended drawings, in which:

Fig. 1

Conjugation efficiency of small cis-methylated conjugative plasmids (n = 3);

Fig. 2

Conjugation efficiency of a large cis-methylated conjugative plasmid (n = 3);

Fig. 3

Comparison of the conjugation efficiency of small conjugative plasmids methylated in cis and trans (n = 3);

Fig. 4

Efficiency of culturing phage lysates, methylated either in trans or in cis (n = 3);

Fig. 5

Efficiency of phage transduction after methylation in trans by a production strain expressing an MTase operon (n = 3).

The present invention demonstrates that it is not necessary to pre-methylate DNA using methyltransferases (MTases) in a production bacterial strain prior to delivery to target bacteria comprising an RM system which would otherwise cleave the introduced DNA and would reduce the efficiency of the transfer. Instead, we surprisingly show that methyltransferases can be directly encoded on nucleic acid that is delivered to a target cell, such as on a phage or conjugative plasmid, and then can be expressed in that target cell to provide protection against restriction modification of the host. This avoids the need for pre-methylation at the production stage of the phage or plasmid.

Furthermore, we demonstrated that DNA methylation using multiple MTases within a production strain also provides significant protection to the DNA during subsequent transfer to a target bacteria. This approach can be directly used in combination with the encoding of MTases in phage or conjugative plasmids if desired.

Furthermore, we compare the methylation of DNA such as plasmid DNA in trans in a cell by a coresident non-conjugative plasmid, with cis methylation. Both approaches are effective in preventing restriction of introduced DNA with highly efficient cis methylation.

In any method or other aspect mentioned herein, where the contacting of a support cell with a target cell is disclosed, in the alternative embodiment, it is provided for the contacting of a population of support cells with a population of target cells.

In a first aspect, the present invention relates to a supporting bacterial cell comprising a conjugative plasmid, the plasmid encoding a methyltransferase enzyme. The data provided here show that inclusion of a methyltransferase enzyme on a conjugative plasmid that is delivered to a target bacteria increases the efficiency of conjugative transfer compared to a plasmid lacking the methyltransferase, in the context of a target bacteria in which this methyltransferase can protect against a host RM system. The invention also provides a population of said support cells, e.g. wherein the population is comprised by a pharmaceutical composition for administration to a human or animal subject to prevent or treat a medical condition.

The invention therefore also relates to a method of delivering plasmid DNA from a support bacterial cell to a target bacterial cell by conjugation, the method comprising bringing the target bacterial cell into contact with the support cell as disclosed here, the A target cell comprising a restriction modification (RM) system that cleaves DNA that is unmethylated at a target methylation site of the methyltransferase enzyme.

In this way, the plasmid DNA transferred by conjugation is protected from cleavage by the RM system in the target cell by the expression, directly in the target cell, of the encoded MTase.

The bringing into contact of the support bacterial cell and the target bacteria can be done by administering the first with a pharmaceutical composition intended to be administered to a human or animal subject to prevent or treat a medical condition, e.g. to an intestinal, pulmonary, skin or renal microbiota. Administration may be oral, inhalation, topical or parenteral (e.g. IV).

Preferably, the plasmid encodes more than one MTase, preferably 2, 3, 4, 5 or even more MTases. Preferably, the plasmid encodes more than one type I MTase. Preferably, the plasmid encodes more than one type II MTase. Preferably, the plasmid encodes more than one type III MTase. Preferably, the plasmid encodes a type I MTase and a type II and/or III MTase.

The plasmid may encode multiple MTases as part of an operon, or the expression of different MTases may be controlled by more than one promoter, for example as part of 2 or more operons. Each nucleotide sequence encoding an MTase may be under the control of a respective promoter.

The plasmid may encode phage DNA or encode an entire phage genome.

Where a bacterial cell or bacteria are mentioned herein in connection with the invention, the invention may instead apply mutatis mutandis to an archaeal cell or archaea.

The plasmid to be transferred by conjugation may itself include methylated DNA to further protect against digestion by restriction enzymes of the target cells. The methylation of the plasmid can be for example by expression of methylase enzymes in the supporting bacterial cell from a plasmid different from the conjugative plasmid, or result from the enzymes of the conjugative plasmid itself, or can result from pre-methylation in a production strain.

The invention therefore relates to a bacterial support cell having a plasmid and/or a genome encoding one or more MTases which provide methylation of said conjugative plasmid DNA in the support cell.

The supporting bacterial cell may be provided in the form of a population of cells such as 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ cells or more.

In a second aspect, the invention relates to a bacteriophage encoding a methyltransferase enzyme, optionally wherein the methyltransferase enzyme is an enzyme that protects against an RM system found in a bacterial species or strain that is pathogenic in humans and animals.

The invention also provides a nucleic acid (e.g. DNA) which encodes such a bacteriophage.

The bacteriophage genome may encode more than one MTase.

The invention also relates to a mixture (also called here cocktail) of at least 2 bacteriophages, each bacteriophage coding for a different methyltransferase enzyme, such as 3, 4, 5 or more bacteriophages, each coding for a different MTase. The invention also relates to a mixture (also referred to herein as a cocktail) of a first and a second bacteriophage, in which each bacteriophage encodes at least one methyltransferase and in which the first bacteriophage encodes a methyltransferase which is not encoded by the second bacteriophage. For example, the first bacteriophage and the second bacteriophage do not encode any MTase in common.

The data provided here show that the inclusion of a methyltransferase enzyme on a bacteriophage that is delivered to a target bacterial cell increases the efficiency of bacteriophage infection compared to a bacteriophage lacking this same methyltransferase, in the context of 'a target bacterial cell in which this methyltransferase can protect against a host RM system.

The invention also relates to a method of delivering a bacteriophage to a target bacterial cell, the method comprising contacting the target cell with a bacteriophage encoding a methyltransferase enzyme as disclosed herein, wherein the target bacteria includes a restriction modification system that cleaves DNA that is unmethylated at a methylation target site of the methyltransferase enzyme.

The data provided here also show that expression of multiple MTases in a production strain can enhance bacteriophage-mediated infection of target bacteria compared to an unmethylated control.

Advantageously, multiple MTases can be used to protect the plasmid, phagemid or phage nucleic acid, from the action of different RM systems within a target bacterial cell. Thus, in one embodiment, the plasmid or phage nucleic acid encodes first and second MTases, the MTases being MTases of different RM systems, e.g. RM systems found together in a single bacterial or archaeal species or strain.

Therefore, in yet another aspect the invention relates to a method of methylation of a bacteriophage DNA or a phagemid DNA or a plasmid DNA, the method comprising:

introducing into a production host bacterium, such as into the genome of a production host bacterium (i) a nucleotide sequence encoding a plurality of methyltransferases, or (ii) two or more nucleotide sequences together encoding a plurality of methyltransferases,

thereby producing an engineered host bacteria expressing at least 2 introduced methyltransferases, suitably which do not occur naturally in the host bacteria cell;

infecting the production host bacteria having altered methylation activity with a bacteriophage particle comprising bacteriophage DNA or phagemid DNA, or transforming the bacteria with a plasmid comprising bacteriophage DNA plasmid, thereby methylating said bacteriophage DNA or phagemid DNA or plasmid DNA; And

producing a bacteriophage particle comprising bacteriophage DNA or phagemid DNA, or producing plasmid DNA, having a methylation pattern produced by methyltransferases in the host bacteria, optionally isolation, further, of the bacteriophage particle comprising bacteriophage DNA or phagemid DNA, or isolation of the plasmid DNA or the plasmid.

Optionally, the method further comprises formulating the isolated bacteriophage, plasmid or DNA with a pharmaceutically acceptable diluent, carrier or excipient to produce a pharmaceutical composition.

The method of methylation of a bacteriophage DNA or a phagemid DNA or a plasmid DNA can also use a bacteriophage or phagemid nucleic acid or a plasmid DNA as described above which encodes itself. even for an MTase, and this process therefore combines methylation of the production strain (provided in trans) and methylation by an MTase or MTases encoded by the DNA which is introduced into the target bacterium (provided in cis).

The invention also relates to a bacteriophage or phagemid or plasmid produced by the above method in the production strain.

Each MTase described herein may be a type 1, type II, or type III MTase. In one example, at least one Type I MTase is used.

An MTase can be provided in cis and/or trans.

When multiple MTases are provided, these may be provided in cis (e.g. on the conjugative plasmid or phage or phagemid that is delivered to a target cell), or in trans (e.g. by a coresident non-conjugative plasmid in a production stem cell or support cell), or both.

In one aspect, the MTase is an Eco0015 MTase, (a Type I MTase) which methylates within the CCANNNNNNNCTTC recognition sequence.

The invention also relates to any MTase that methylates within the recognition sequence CCANNNNNNCTTC

In one aspect, the MTase is an EcoK1 MTase, (type I) which methylates within the recognition sequence AACNNNNNNGTGC

The invention also relates to any MTase that methylates within the sequence AACNNNNNNGTGC

Other methylases that may be encoded on the phage, conjugative plasmid or production strain include M. EcoA1, M. EcoAO831, M.EcoMII, M.EcoMIII, M1 M2 Eco31I, M.Eco1167.

The invention also relates to any MTase that methylates within a subsequent sequence. These sequences are, for example, associated with MTase as listed below: M.EcoAI G A GNNNNNNNG T CA M.EcoAO83I GGANNNNNNNNATGC M.EcoMII RT A CNNNNG T G M.EcoMIII AAAGNNNNGTT M1 M2 Eco31I GG T C TC M.Eco1167 YTC A NNNNNNGTTY M.EcoRII C C WGG

In particular, we identified the prevalence of these sites as follows: REBASE identifier Recognition sequence* Kind Prevalence in E. coli** EcoMII RT A CNNNNG T G I 147 EcoAI G A GNNNNNNNG T CA I 48 EcoKI A A CNNNNNNNG T GC I 38 EcoAO83I GGANNNNNNNNATGC I 34 EcoMIII AA A GNNNNG T T I 157 Eco0015 CCANNNNNNCTTC I 55 Eco1167 YTCANNNNNNGTTY I 29 M1.Eco31I GG T CTC II 177

* recognition sequence with methylated A in bold and T corresponding to the methylated A on the underlined complementary strand (in the absence of indication: the methylated bases have not been determined)

** prevalence in a collection of 932 clinical isolates of E. coli in total

The most prevalent RM systems found in the genome sequence of clinical isolates of E. coli were assigned an identifier based on a minimum 99.5% amino acid identity with the sequences described in REBASE (http://rebase.neb.com/rebase/rebase.html)

In any aspect, the methyltransferase may be a methyltransferase from a pathogenic bacterial strain or species.

In any aspect, the methyltransferase may be a methyltransferase from a bacteria associated with bacterial corrosion or biofouling.

The methyltransferase may be a methyltransferase that protects against MR type I, type II, or type III systems, and optionally multiple systems, such as type I and type II, type I and type III, or type II and III, or all three.

The methyltransferase may be a methyltransferase that protects against an RM system present in any of the bacteria Staphylococcus, Streptococcus, Pseudomonas, Salmonella, Listeria, E coli, Desulfovibrio or Clostridium . For example, the methyltransferase may be a methyltransferase that protects against an RM system found in E coli . For example, the methyltransferase may be a methyltransferase that protects against an RM system found in C difficile . For example, the methyltransferase may be a methyltransferase that protects against an RM system found in K pneumoniae . For example, the methyltransferase may be a methyltransferase that protects against an RM system found in P aeruginosa . For example, the methyltransferase may be a methyltransferase that protects against an RM system found in S typhimurium . For example, the methyltransferase may be a methyltransferase that protects against an RM system found in H pylori .

The MTase may be any MTase that protects against an RM system found in a bacterial species disclosed in Table 1 of WO 2017/211753 A1, which listing is specifically incorporated herein by reference.

The MTase may be an MTase from Enterobacteriaceae such as E. coli, Salmonella, Yersinia pestis, Klebsiella, Shigella, Proteus, Enterobacter, Serratia and Citrobacter .

An MTase can be an orphan or promiscuous MTase.

When more than one MTase is encoded, it may be a combination of a promiscuous MTase and a specific non-promiscuous MTase.

The bacteriophage referred to herein may be any bacteriophage suitable for targeting a target cell that carries an RM system that cleaves unmethylated DNA, preferably DNA viruses.

The Bacteriophages mentioned herein may be any phage of a genus selected from Dhakavirus , Gaprivervirus , Gelderlandvirus , Jiaodavirus , Karamvirus , Krischvirus , Moonvirus , Mosigvirus , Schizotequatrovirus , Slopekvirus and Tequatrovirus . Each phage mentioned here may be an Enterobacteriaceae phage, E coli phage, Myoviridae phage, Tevenvirinae phage, Tequatrovirus phage, Caudovirales phage, adeno-associated viruses (AAV), herpes simplex viruses, retroviruses or lentiviruses. For example, each virus or phage mentioned herein may be of a genus selected from Dhakavirus , Gaprivervirus , Gelderlandvirus , Jiaodavirus , Karamvirus , Krischvirus , Moonvirus , Mosigvirus , Schizotequatrovirus , Slopekvirus , and Tequatrovirus . Each phage mentioned here may be a Klebsiella virus (e.g. Klebsiella phage PMBT1, Klebsiella phage PKO111, Klebsiella phage phi KpNIH-6, Klebsiella phage Miro, Klebsiella phage vB_KpnM_Kp_V477, Klebsiella phage KP27, Klebsiella phage KP15, Klebsiella phage KP1 or Klebsiella phage JD18), an Acinetobacter phage (e.g. Acinetobacter virus 133), an Aeromonas phage (e.g. Aeromonas virus 65 or Aeromonas virus Aeh1) , an Escherichia phage (e.g. Escherichia virus RB16, Escherichia virus RB32 or Escherichia virus RB43) or a Pseudomonas phage (e.g. Pseudomonas virus 42). Each phage mentioned here may be a Tevenvirinae phage, e.g. a phage selected from Table D. A phagemid mentioned herein may be a modified version of the genome of any phage disclosed in this paragraph.

Cocktails (mixtures) of 2 or more bacteriophages can be used in which each bacteriophage of the same type encodes a different MTase enzyme necessary for protection in a target host cell.

A cocktail or mixture as mentioned herein may be a population of phages, the population consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or more than 10) different types of bacteriophages or of phagemids, each bacteriophage/phagemid being in accordance with the invention.

Alternatively, a bacteriophage cocktail may also include a phage having a different bacteriophage backbone as well as a different MTase - thus, the cocktail could, for example, be bacteriophage P1 and M13, where the P1 encodes an MTase that does not is not encoded by the M13, where the M13 encodes an MTase which is (or is not) encoded by the P1; or where the M13 encodes an MTase which is not encoded by the P1, where the P1 encodes an MTase which is (or is not) encoded by the M13.

In one embodiment, the bacteriophages are specific to a single strain or species of bacteria. Here, those skilled in the art will understand that "specific to" refers to the host infectivity profile of each phage.

In another embodiment, bacteriophages which are capable of infecting two or more bacterial strains or two or more species of bacteria may be used.

In one embodiment, each bacteriophage, plasmid or phagemid is intended for use in humans or animals in vivo , e.g. for use as a medicine.

In one embodiment, each bacteriophage, plasmid or phagemid is intended for use in an environment, e.g. for use in remediating biofouling or microbial corrosion of a surface (e.g. a metal surface).

In one embodiment, each bacteriophage, plasmid or phagemid is used ex vivo or in vitro .

The phage as disclosed herein may be P1 phage, M13 phage, A phage, T4 phage, PhiC2 phage, PhiCD27 phage, PhiNMl phage, Bc431v3 phage, Phi10 phage, Phi25 phage, Phi151, an A511 type phage, a B054 phage, an 01761 type phage or a Campylobacter phage, optionally NCTC12676 or NCTC12677. The phage can be a lambda phage. The phage may be any phage of a genus selected from Dhakavirus , Gaprivervirus , Gelderlandvirus , Jiaodavirus , Karamvirus , Krischvirus , Moonvirus , Mosigvirus , Schizotequatrovirus , Slopekvirus and Tequatrovirus , such as those disclosed herein .

The phagemid as disclosed herein may be a P1 phagemid, an M13 phagemid, an A phagemid, a T4 phagemid, a PhiC2 phagemid, a PhiCD27 phagemid, a PhiNMl phagemid, a Bc431v3 phagemid, a Phi10 phagemid, a Phi25 phagemid, a phagemid Phi151, an A511 type phagemid, a B054 phagemid, a 01761 type phagemid, a Campylobacter phagemid, optionally NCTC12676 or NCTC12677. In one example, the phagemid is a lambda phagemid. The phagemid may be derived from any phage of a genus selected from Dhakavirus , Gaprivervirus , Gelderlandvirus , Jiaodavirus , Karamvirus , Krischvirus , Moonvirus , Mosigvirus , Schizotequatrovirus , Slopekvirus and Tequatrovirus , such as those disclosed herein .

A phagemid may encode a non-self-replicating transducing particle, as is familiar to those skilled in the art.

A conjugative plasmid as disclosed herein is a plasmid which is transferred by bacterial conjugation. Conjugative plasmids include at least one transfer origin, oriT, and may include one or more or all of a set of transfer (tra) genes required for conjugation. These tra gene functions can alternatively be provided in whole or in part by the support cell comprising the plasmid.

In a preferred embodiment, all necessary tra genes required for conjugation are included on the plasmid.

The supporting bacterial cell comprising the conjugative plasmid may be any suitable bacteria, but is preferably a bacteria which is not pathogenic in humans or animals. The bacterial species may be any species disclosed herein, e.g. E. coli or a species of Bacteriodies. The supporting bacterial cell may be, for example, a Bacteriodes, Escherichia, Salmonella, Clostridium, Bifidobacterium , Lactobacillus, Pseudomonas, Caulobacter , Listeria, Proteus or Streptococcus cell.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in combination with CRISPR-associated genes constitute the CRISPR-Cas system , which confers adaptive immunity to many bacteria and most archaea. CRISPR-mediated immunization occurs through DNA integration of invasive genetic elements such as plasmids and bacteriophages that can be used to prevent future infections by invaders containing the same sequence. CRISPR-Cas systems consist of CRISPR arrays of short DNA “repeats” spaced by hypervariable “spacer” sequences and a set of flanking cas genes. In any aspect of the invention, the nucleic acid which is delivered to the target cell may comprise a CRISPR template, suitably a template which comprises at least one spacer substantially complementary to a target DNA sequence in the target bacteria. . In this way, the target DNA sequence of the target bacteria can be modified by the CRISPR-cas system using the spacer to specifically target the target sequence. Methylation of the DNA encoding the template – as well as all other components of the CRISPR-cas system that are also delivered to the target cell – is protected from restriction cleavage and will therefore function more efficiently within the target cell. The CRISPR array is also referred to herein as target cell modification CRISPR array.

The CRISPR template can be part of the bacteriophage nucleic acid, phagemid nucleic acid, or conjugative plasmid.

The template suitably includes a spacer sequence and repeats encoding a crRNA, the crRNA comprising a sequence that hybridizes to a target DNA of a target cell to guide a Cas enzyme to the target.

The modification of the target gene sequence that results from delivery of a CRISPR template into the target bacteria may include cleavage of one or both strands of the target cellular DNA at the target sequence, causing inactivation of gene expression, directly or indirectly, or can lead to the destruction of the cell.

In one aspect, the CRISPR array is a nucleic acid molecule that includes at least two repeat sequences and at least one spacer sequence, one of the two repeat sequences being linked to the 5' end of the spacer sequence and the other of the two repeat sequences is linked to the 3' end of the spacer sequence. The CRISPR array may include 2 or more repeat spacer sequences, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25 or more repeat spacer sequences, suitably all spacers in said array being flanked on both the 5' end and the 3' end by a repeat sequence .

Other components of the CRISPR-cas system may be included in (encoded by) the bacteriophage nucleic acid, phagemid nucleic acid, or conjugative plasmid, and these include one or more of

(i) a cas endonuclease,

(ii) a tracrRNA sequence or a DNA sequence expressing a tracrRNA sequence.

Cas endonuclease functionality may alternatively be provided naturally from within the target bacteria.

When provided as part of the bacteriophage, phagemid or plasmid, the cas endonuclease may be a cas type I, type II, type III, type IV, type V or type VI nuclease, such as cas3 or cas9 or cpf1.

The spacer sequence is complementary to a target DNA sequence in the genome of the target cell, and may be fully (100%) complementary or substantially complementary (e.g. at least about 70% complementary (e.g. about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the target DNA. The spacer sequence may have one, two, three, four or five mismatches to the target DNA. The mismatches may be contiguous or non-contiguous. The spacer sequence may have complete complementarity or substantial complementarity over a region of a target nucleotide sequence that has a length of at least about 20 nucleotides to about 100 nucleotides.

The supporting bacterial cell, bacteriophage, phagemid or plasmid disclosed herein preferably encodes an MTase and a payload, e.g. a CRISPR network. For example, the payload and MTase are encoded on the same DNA.

The supplied nucleic acid may encode a guided nuclease (optionally a Cas) and/or a guide RNA and/or the supplied nucleic acid comprises a CRISPR template to produce a crRNA in the target cell. The guided nuclease may be a Cas nuclease (e.g. a Cas Type I, II, III, IV, V or VI nuclease, e.g. a Cas9, a Cas3, a Cas12 or a Cas13). The guided nuclease may be a TALEN, a zinc finger nuclease or a meganuclease.

The supplied nucleic acid may comprise or consist of 1 to 10 kb, e.g. 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 kb of DNA. For example, the nucleic acid provided comprises a CRISPR template (and/or a nucleotide sequence coding for a guide RNA, such as a single guide RNA) and optionally one or more nucleotide sequence(s) each coding for a respective case.

The supplied nucleic acid may encode a CRISPR Cascade protein (e.g. Cas A, B, C, D or E).

The nucleic acid provided may encode a crRNA. Heterologous DNA can encode single guide RNA (sgRNA). Heterologous DNA may encode a tracrRNA.

Here are the preferred aspects of the CRISPR-cas system components:

The Case may be any Case (e.g., a Case2, 3, 4, 5, or 6) of a Type I system. In one embodiment, the Case may be merged or conjugated to a fragment that is usable to augment or reduce transcription of a gene comprising the target proto-spacer sequence. For example, the nucleic acid encoding Cas that is introduced into a cell may comprise a nucleotide sequence encoding the fragment, where Cas and the fragment are expressed in the host cell as a fusion protein. In one embodiment, the Cas is N-terminal of the fragment; in another embodiment, it is C-terminal of the fragment.

Optionally, one or more Cascade protein(s) may be provided by or to a target cell. For example, a first Cas (C1) and/or a second Cas (C2) may be provided and the Cascade protein(s) is(are) related to the C1 or C2, which is a Case3. For example, a first Cas (C1) and/or a second Cas (C2) are provided and Cas1 or Cas2 is a Cas3 which is related to Cascade proteins encoded by the cell.

Optionally, the Cascade proteins include or consist of cas5 (casD, csy2), cas6 (cas6f, cse3, casE), cas7 (csc2, csy3, cse4, casC) and cas8 (casA, cas8a1, cas8b1, cas8c, cas10d, cas8e, cse1, cas8f, csy1).

Optionally, provided nucleic acid is provided, comprising a promoter and a Cas3-encoding or crRNA-encoding sequence that are spaced no more than 150, 100, 50, 40, 30, 20 or 10 bp apart, and are p. ex. spaced 30-45, or 30-40, or 39 or about 39 bp. Optionally here, a ribosome binding site and the sequence coding for Cas3 or coding for crRNA are spaced no more than 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 4 or 3 bp, and are p. ex. separated by 10 to 5, 6 or approximately 6 bp.

In one example, a promoter mentioned herein is in combination with a Shine-Dalgarno sequence comprising the sequence 5'-aaagaggagaaa-3' (SEQ ID NO: 1) or a ribosome binding site homolog thereof. Optionally, the promoter has an Anderson score (AS) of AS ≥ 0.5; or an Anderson score (AS) of 0.5 > AS > 0.1; or an Anderson score (AS) of ≤ 0.1.

Optionally, the nucleic acid provided lacks a nucleotide sequence encoding one, more or all of Cas1, Cas2, Cas4, Cas6 (optionally Cas6f), Cas7 and Cas 8 (optionally Cas8f). Optionally, the DNA lacks a sequence coding for a Cas6 (optionally a Cas6f). Optionally, the DNA comprises (optionally in the 5' to 3' direction) a nucleotide sequence encoding one, more or all of Cas11, Cas7 and Cas8a1. Optionally, the DNA comprises a nucleotide sequence encoding Cas3' and/or Cas3''. In one embodiment, the DNA includes nucleotide sequences (in the 5' to 3' direction) that encode Cas3 (e.g., Cas3' and/or Cas3''), Cas11, Cas7, and Cas8a1.

Optionally, a nucleotide sequence encoding Cas6 is located between the Cas3 sequence(s) and the Cas11 sequence.

Optionally, the nucleic acid provided comprises a Type IA CRISPR matrix or one or more nucleotide sequence(s) coding for one or more unique guide RNA(s) (gRNA(s)), the matrix and each gRNA comprising a repeat sequence which is related to a Cas3. Thus, the template is usable in a host cell when the hybrid DNA has been introduced into the cell for the production of guide RNAs, where the guide RNAs can be used with Cas and Cascade proteins to target and modify (e.g. , cut) a target nucleotide sequence in the host cell, thus optionally killing the cell. Likewise, the unique guide RNAs encoded by the nucleic acid provided in one embodiment are for use with Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the host cell, thereby killing optionally the cell.

Optionally, a target cell comprises a Type IA CRISPR array that is related to Cas3 (C1 or C2). Optionally, a target cell comprises an endogenous CRISPR/Cas system of Type IB, C, U, D, E or F. Optionally, the nucleic acid provided comprises (optionally in the 5' to 3' direction) a nucleotide sequence coding for one, more or all of Cas8b1, Cas7 and Cas5. In one embodiment, the provided nucleic acid comprises nucleotide sequences (in the 5' to 3' direction) which encode Cas3, Cas8b1, Cas7 and Cas5. Optionally, a nucleotide sequence encoding Cas6 is located between the Cas3 sequence(s) and the Cas8b1 sequence. Optionally, the nucleic acid provided comprises a Type IB CRISPR matrix or one or more nucleotide sequence(s) coding for one or more unique guide RNA(s) (gRNA(s)), the matrix and each gRNA comprising a repeat sequence which is related to Cas3. Thus, the matrix can be used in a host cell when the nucleic acid provided has been introduced into the cell for the production of guide RNAs, the guide RNAs being usable with Cas and Cascade proteins to target and modify (eg. , cut) a target nucleotide sequence in the host cell, thus optionally killing the host cell. Likewise, the unique guide RNAs encoded by the nucleic acid provided in one embodiment are usable with Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, thereby optionally killing the cell.

Optionally, the target cell comprises a Type IB CRISPR template which is related to Cas3.

Optionally, the target cell comprises an endogenous CRISPR/Cas system of Type IA, C, U, D, E or F. Optionally, the nucleic acid provided comprises (optionally in the 5' to 3' direction) a nucleotide sequence coding for one, more or all of Cas5, Cas8c and Cas7. In one embodiment, the provided nucleic acid comprises nucleotide sequences (in the 5' to 3' direction) which encode Cas3, Cas5, Cas8c and Cas7. Optionally, a nucleotide sequence encoding Cas6 is located between the Cas3 sequence(s) and the Cas5 sequence. Optionally, the nucleic acid provided comprises a Type IC CRISPR matrix or one or more nucleotide sequence(s) coding for one or more unique guide RNA(s) (gRNA(s)), the matrix and each gRNA comprising a repeat sequence which is related to Cas3. Thus, the matrix can be used in a host cell when the nucleic acid provided has been introduced into the cell for the production of guide RNAs, the guide RNAs being usable with Cas and Cascade proteins to target and modify (eg. , cut) a target nucleotide sequence in the cell, thus optionally killing the cell. Likewise, the unique guide RNAs encoded by the nucleic acid provided in one embodiment are usable with Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, thereby optionally killing the cell.

Optionally, the target cell comprises a Type IC CRISPR template which is related to Cas3. Optionally, the target cell comprises an endogenous CRISPR/Cas system of Type IA, B, U, D, E or F. Optionally, the nucleic acid provided comprises (optionally in the 5' to 3' direction) a nucleotide sequence coding for one, more or all of Cas8U2, Cas7, Cas5 and Cas6. In one embodiment, the provided nucleic acid comprises nucleotide sequences (in the 5' to 3' direction) which encode Cas3, Cas8U2, Cas7, Cas5 and Cas6. Optionally, a nucleotide sequence encoding Cas6 is located between the Cas3 sequence(s) and the Cas8U2 sequence.

Optionally, the nucleic acid provided comprises a Type IU CRISPR matrix or one or more nucleotide sequence(s) coding for one or more unique guide RNA(s) (gRNA(s)), the matrix and each gRNA comprising a repeat sequence which is related to Cas3. Thus, the matrix can be used in a host cell when the nucleic acid provided has been introduced into the cell for the production of guide RNAs, the guide RNAs being usable with Cas and Cascade proteins to target and modify (eg. , cut) a target nucleotide sequence in the cell, thus optionally killing the cell. Likewise, the unique guide RNAs encoded by the vector in one embodiment are operable with Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, thereby optionally killing the cell.

Optionally, the target cell comprises a Type IU CRISPR template which is related to Cas3. Optionally, the host cell comprises an endogenous CRISPR/Cas system of Type IA, B, C, D, E or F. Optionally, the vector comprises (optionally in the 5' to 3' direction) a nucleotide sequence encoding one, more or all of Cas10d, Cas7 and Cas5. Optionally, the nucleic acid provided comprises a nucleotide sequence encoding Cas3' and/or Cas3''. In one embodiment, the provided nucleic acid comprises nucleotide sequences (in the 5' to 3' direction) which encode Cas3, Cas10d, Cas7 and Cas5. Optionally, a nucleotide sequence encoding Cas6 is located between the Cas3 sequence(s) and the Cas10d sequence. Optionally, the nucleic acid provided comprises a Type ID CRISPR matrix or one or more nucleotide sequence(s) coding for one or more unique guide RNA(s) (gRNA(s)), the matrix and each gRNA comprising a repeat sequence which is related to Cas3. Thus, the template is usable in a cell when the vector has been introduced into the cell for the production of guide RNAs, where the guide RNAs can be used with Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence into the cell, thus optionally killing the cell. Likewise, the unique guide RNAs encoded by the nucleic acid provided in one embodiment are usable with Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, thereby optionally killing the cell.

Optionally, the target cell comprises a Type ID CRISPR template which is related to Cas3.

Optionally, the target cell comprises an endogenous CRISPR/Cas system of Type IA, B, C, U, E or F.

Optionally, the nucleic acid provided comprises (optionally in the 5' to 3' direction) a nucleotide sequence encoding one, more or all of Cas8e, Cas11, Cas7, Cas5 and Cas6. In one embodiment, the nucleic acid provided comprises nucleotide sequences (in the 5' to 3' direction) which encode Cas3, Cas8e, Cas11, Cas7, Cas5 and Cas 6. Optionally, a nucleotide sequence encoding Cas6 is located between the Cas3 sequence(s) and the Cas11 sequence. Optionally, the nucleic acid provided comprises a Type IE CRISPR matrix or one or more nucleotide sequence(s) coding for one or more unique guide RNA(s) (gRNA(s)), the matrix and each gRNA comprising a repeat sequence which is related to Cas3. Thus, the template is usable in a host cell when the vector has been introduced into the cell for the production of guide RNAs, where the guide RNAs can be used with Cas and Cascade proteins to target and modify (e.g., cut ) a target nucleotide sequence in the cell, thus optionally killing the cell. Likewise, the unique guide RNAs encoded by the nucleic acid provided in one embodiment are usable with Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, thereby optionally killing the cell.

Optionally, the target cell comprises a Type IE CRISPR template which is related to Cas3.

Optionally, the target cell comprises an endogenous CRISPR/Cas system of Type IA, B, C, D, U or F.

Optionally, the nucleic acid provided comprises (optionally in the 5' to 3' direction) a nucleotide sequence encoding one, more or all of Cas8f, Cas5, Cas7 and Cas6f. In one embodiment, the provided nucleic acid comprises nucleotide sequences (in the 5' to 3' direction) which encode Cas3, Cas8f, Cas5, Cas7 and Cas6f. Optionally, a nucleotide sequence encoding Cas6 is located between the Cas3 sequence(s) and the Cas8f sequence. Optionally, the nucleic acid provided comprises a Type IF CRISPR matrix or one or more nucleotide sequence(s) coding for one or more unique guide RNA(s) (gRNA(s)), the matrix and each gRNA comprising a repeat sequence which is related to Cas3. Thus, the template is usable in a cell when the vector has been introduced into the cell for the production of guide RNAs, where the guide RNAs can be used with Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence into the cell, thus optionally killing the cell. Likewise, the unique guide RNAs encoded by the nucleic acid provided in one embodiment are usable with Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, thereby optionally killing the cell.

Optionally, the cell comprises a Type IF CRISPR template which is related to Cas3

Optionally, the cell comprises an endogenous CRISPR/Cas system of Type IA, B, C, D, U or E.

Optionally, Cas and Cascade are Type IA Cas and Cascade proteins.

Optionally, Cas and Cascade are Type IB Cas and Cascade proteins.

Optionally, Cas and Cascade are Type IC Cas and Cascade proteins.

Optionally, Cas and Cascade are Type ID Cas and Cascade proteins.

Optionally, Cas and Cascade are Type IE Cas and Cascade proteins.

Optionally, Cas and Cascade are Type IF Cas and Cascade proteins.

Optionally, Cas and Cascade are Type IU Cas and Cascade proteins.

Optionally, the Cas and Cascade are E coli Cas and Cascade proteins (optionally Type IE or IF), the E coli optionally being an ESBL-producing E. coli or E. coli ST131-O25b:H4.

Optionally, Cas and Cascade are Cas and Cascade proteins from Clostridium (e.g., from C difficile), optionally from C difficile resistant to one or more antibiotics selected from aminoglycosides, lincomycin, tetracyclines, erythromycin, clindamycin, penicillins, cephalosporins and fluoroquinolones.

Optionally, Cas and Cascade are Cas and Cascade proteins from Pseudomonas aeruginosa, optionally from P aeruginosa resistant to one or more antibiotics selected from carbapenems, aminoglycosides, cefepime, ceftazidime, fluoroquinolones, piperacillin and tazobactam.

Optionally, Cas and Cascade are Cas and Cascade proteins from Klebsiella pneumoniae (e.g., carbapenem-resistant Klebsiella pneumoniae or extended-spectrum beta-lactamase (ESBL)-producing K pneumoniae).

Optionally, Cas and Cascade are Cas and Cascade proteins from E coli, C difficile, P aeruginosa, K pneumoniae, P furiosus or B halodurans.

Optionally, each crRNA or gRNA comprises a spacer sequence which is capable of hybridizing with a proto-spacer nucleotide sequence of the cell, the proto-spacer sequence being adjacent to a PAM (from the English "protospacer adjacent motif”, or “motif adjacent to the proto-spacer”), the PAM being related to C1 or C2, C1 or C2 being a Cas nuclease, p. e.g., a Case3. Thus, the spacer hybridizes with the proto-spacer to guide Cas3 towards the proto-spacer. Optionally, Cas3 cuts the proto-spacer, e.g. ex. using the exo- and/or endonuclease activity of Cas3. Optionally, Cas3 removes a plurality (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides from the protospacer.

The invention also relates to pharmaceutical compositions comprising the bacteriophage, phagemid or carrier cells as disclosed herein in combination with a pharmaceutically acceptable diluent, carrier or excipient. Suitable carriers and excipients are well known and include suitably sterilized water, saline solutions and buffered saline solutions. In some aspects, when delivered to the gastrointestinal tract, the carrier cell or phage delivered is suitably protected from acid by an enteric coating, or other coating that can prevent an adverse effect on the cell or phage. phage in the gastric environment such as the stomach.

Any composition (e.g. a pharmaceutical composition) mentioned herein may be understood by a medical container or a sterile container, e.g. a syringe, IV bag, auto-injector pen or vial.

When used, the bacteriophage can be freeze-dried prior to delivery and then reconstituted before use with a suitable diluent.

The provision of the bacteriophage, phagemid or support cell comprising a conjugative plasmid as disclosed herein may be part of a method for preventing or treating a human or animal disease, optionally in combination with a targeted CRISPR-CAS modification of a target sequence within the target bacteria.

In one embodiment, modification at the target DNA of the target bacteria results in destruction or attenuation of the target bacteria. When the target bacteria is pathogenic, the modification then provides prevention or treatment of a pathogenic bacterial disease directly by said destruction or attenuation of the pathogenic bacteria.

Consequently, the invention relates to a method for preventing or treating a disease caused by a pathogenic target bacteria in humans or animals, the method comprising the provision of a bacteriophage, a phagemid or a supporting bacterial cell comprising a conjugative plasmid as disclosed herein, or a pharmaceutical composition as disclosed herein, to the human or animal in need thereof, and a bacteriophage, a phagemid or a supporting bacterial cell according to such as disclosed ( e) here, or a pharmaceutical composition as disclosed herein, intended for use in a process for preventing or treating a disease caused by a pathogenic target bacteria in humans or animals, the process comprising providing of the bacteriophage, the phagemid or the supporting bacterial cell or the pharmaceutical composition to the human or animal in need.

The modification may also provide indirect treatment or prevention of disease, in the event that bacteria present in a host negatively affect the host's response to diseases other than bacterial disease. For example, patients' responses to cancer treatments have been shown to be influenced by the composition of their microbiome, see Vancheswaran Gopalakrishnan et al, Cancer Cell. April 9, 2018; 33(4): 570–580.

Therefore, the present invention also relates to a method for preventing or treating a disease, the method comprising providing a bacteriophage, phagemid or bacterial support cell comprising a conjugative plasmid as disclosed herein, or a composition pharmaceutical disclosed herein, to a human or animal in need thereof, the human or animal comprising a target bacteria having a target sequence the modification of which results in improved responses to cancer or cancer immunotherapy.

For example, target bacteria may be Salmonella, Clostridium, Bifidobacterium , Lactobacillus, Escherichia, Pseudomonas, Caulobacter , Listeria, Proteus and Streptococcus .

The target bacteria may be any bacteria listed in Table 1 of WO 2017/211753 A1, which list is specifically incorporated herein by reference.

The bacteriophage, phagemid and carrier cells and pharmaceutical compositions disclosed herein may also be used in methods in combination with other medical treatments, including, but not limited to,

(i) anticancer agents,

(ii) antibiotics and

(iii) bacterial cells which are used to repopulate the human or animal microbiota.

Examples of suitable medications are provided in Table E

The delivery of a bacteriophage, a phagemid and support cells and pharmaceutical compositions as disclosed can be done by any suitable delivery route, such as intravenous, intradermal, subcutaneous, intramuscular, topical, by inhalation or intranasal intake, or oral intake.

The present invention can be used in the treatment of diseases or conditions such as the following:

(a) A neurodegenerative disease or condition;

(b) A disease or condition of the brain;

(c) A disease or condition of the CNS;

(d) Loss or impairment of memory;

(e) A cardiac or cardiovascular disease or condition, e.g. a heart attack, stroke or atrial fibrillation;

(f) A liver disease or condition;

(g) A disease or condition of the kidneys, e.g. chronic kidney disease (CKD);

(h) A disease or condition of the pancreas;

(i) A lung disease or condition, e.g. cystic fibrosis or COPD;

(j) A gastrointestinal disease or condition;

(k) A disease or condition of the throat or oral cavity;

(l) An eye disease or condition;

(m) A genital disease or condition, e.g. a vaginal, labial, penile or scrotal disease or condition;

(n) A sexually transmitted disease or condition, e.g. gonorrhea, HIV infection, syphilis or chlamydia infection;

(o) A disease or condition of the ear;

(p) A skin disease or condition;

(q) A heart disease or condition;

(r) A nasal disease or condition;

(s) A hematological disease or condition, e.g. anemia, e.g. anemia resulting from a chronic illness or cancer;

(t) A viral infection;

(u) A pathogenic bacterial infection;

(v) Cancer;

(w) An autoimmune disease or condition, e.g. the LED;

(x) An inflammatory disease or condition, for example rheumatoid arthritis, psoriasis, eczema, asthma, ulcerative colitis, colitis, Crohn's disease or IBD;

(y) Autism;

(z) ADHD;

(aa) Bipolar disorder;

(bb) ALS [Amyotrophic Lateral Sclerosis];

(cc) Osteoarthritis;

(dd) A congenital or developmental defect or condition;

(ee) A miscarriage;

(ff) A blood clotting disease;

(gg) Bronchitis;

(hh) dry or wet AMD;

(ii) Neovascularization (e.g. from a tumor or in the eye);

(dd) Common cold;

(kk) Epilepsy;

(ll) Fibrosis, e.g. hepatic or pulmonary fibrosis;

(mm) A fungal disease or condition, e.g. thrush;

(nn) A metabolic disease or condition, e.g. obesity, anorexia, diabetes, Type I or Type II diabetes;

(oo) Ulcer(s), e.g. gastric ulceration or skin ulceration;

(pp) Dry skin;

(qq) Sjögren syndrome;

(rr) Cytokine shock;

(ss) Deafness, hearing loss or impairment;

(tt) Slow or rapid metabolism (i.e. slower or faster than average for the subject's weight, sex and age);

(uu) Conception disorder, e.g. infertility or low fertility;

(vv) Jaundice;

(ww) Rash;

(xx) Kawasaki disease;

(yy) Lyme disease;

(zz) An allergy, e.g. nuts, grass, pollen, mites, fur or dander from cats or dogs;

(aaa) Malaria, typhoid fever, tuberculosis or cholera;

(bbb) Depression;

(ccc) Mental retardation;

(ddd) Microcephaly;

(eee) Malnutrition;

(fff) Conjunctivitis;

(ggg) Pneumonia;

(hhh) Pulmonary embolism;

(iii) Pulmonary hypertension;

(jjj) Bone disorder;

(kkk) Sepsis or septic shock;

(lll) Sinusitis;

(mmm) Stress (e.g. work stress);

(nnn) Thalassemia, anemia, von Willebrand disease or hemophilia;

(ooo) Shingles or cold sore;

(dpi) Menstruation;

(qqq) Low sperm count.

NEURODEGENERATIVE OR CNS DISEASES OR CONDITIONS TO TREAT OR PREVENT

In one example, a neurodegenerative or CNS disease or condition is selected from the group consisting of Alzheimer's disease, geriopsychosis, Down syndrome, Parkinson's disease, Creutzfeldt-Jakob disease, diabetic neuropathy, Parkinson's syndrome, Huntington's disease, Machado-Joseph disease and amyotrophic lateral sclerosis. For example, the disease is Alzheimer's disease. For example, the disease is Parkinson's syndrome.

In one example, where the method of the invention is performed on a human or animal subject to treat a CNS or neurodegenerative disease or condition, the method causes downregulation of Treg cells in the subject, thereby promoting entry of systemic macrophages derived from monocytes and/or Treg cells through the choroid plexus into the brain of the subject, whereby the disease or condition (e.g. Alzheimer's disease) is treated, prevented or its progression is reduced. In one embodiment, the method causes an increase in IFN-gamma in the CNS system (e.g. in the brain and/or CerebroSpinal Fluid or CSF) of the subject. In one example, the method restores the nerve fiber and/or reduces the progression of nerve fiber damage. In one example, the method restores nerve myelin and/or reduces the progression of nerve myelin damage. In one example, the method of the invention treats or prevents a disease or condition disclosed in WO2015136541 and/or the method may be used with any method disclosed in WO2015136541 (the disclosure of this document is incorporated herein by reference in its entirety, e.g., to provide the description of potential methods, diseases, conditions and therapeutic agents that may be administered to the subject to effect the treatment and/or prevention of CNS and neurodegenerative diseases and conditions, e.g., agents such as immune checkpoint inhibitors, e.g. , anti-PD-1, anti-PD-L1, anti-TIM3 or other antibodies disclosed therein).

CANCERS TO TREAT OR PREVENT

Cancers that can be treated include tumors that are not vascularized, or not substantially vascularized, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematological tumors, e.g. leukemias and lymphomas) or may include solid tumors. Types of cancers to be treated with the invention include, but are not limited to, carcinoma, blastoma and sarcoma, and certain lymphoid leukemias or malignancies, benign and malignant tumors, and malignancies e.g. sarcomas, carcinomas and melanomas . Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), leukemias chronic (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), myelomas multiple, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Solid tumors are abnormal tissue masses that usually do not contain a cyst or fluid area. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, malignant lymphopathy, cancer pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma , papillary thyroid carcinoma, pheochromocytomas, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma (hepatoma), cholangiocarcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor , seminoma, bladder carcinoma, melanoma and CNS tumors (such as glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also called glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma , craniopharyogioma with neuroma, ependymoma, pineaioma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma and brain metastases).

AUTOIMMUNE DISEASES TO TREAT OR PREVENT

Acute Disseminated Encephalomyelitis (ADEM or Acute Disseminated Encephalomyelitis)

Acute necrotizing hemorrhagic leukoencephalitis

Addison's disease

Agammaglobulinemia

Alopecia Areata (Alopecia areata)

Amyloidosis

Ankylosing spondylitis

Anti-GBM/Anti-TBM nephritis

Antiphospholipid syndrome (APS)

Autoimmune angioedema

Autoimmune aplastic anemia

Autoimmune neurovegetative dystonia

Autoimmune hepatitis

Autoimmune hyperlipidemia

Autoimmune immunodeficiency

Autoimmune inner ear disease (AIED)

Autoimmune myocarditis

Autoimmune oophoritis

Autoimmune pancreatitis

Autoimmune retinopathy

Autoimmune Thrombocytopenic Purpura (ATP or Autoimmune Thrombocytopenic Purpura)

Autoimmune thyroid disease

Autoimmune urticaria

Axonal and neuronal neuropathies

Balo's disease

Behcet's disease

Bullous pemphigoid

Cardiomyopathy

Castleman disease

Celiac disease

Chagas disease

Chronic fatigue syndrome

Chronic inflammatory demyelinating polyneuropathy (CIDP)

Chronic recurrent multifocal ostomyelitis (CRMO)

Churg-Strauss syndrome

Cicatricial pemphigoid/benign mucosal pemphigoid

Crohn's disease

Cogan syndrome

Cold agglutinin disease

Congenital heart block

Myocarditis in Coxsackie

CREST syndrome

Mixed essential cryoglobulinemia

Demyelinating neuropathies

Dermatitis herpetiformis

Dermatomyositis

Devic's disease (neuromyelitis optica)

Discoid lupus

Dressler syndrome

Endometriosis

Eosinophilic esophagitis

Eosinophilic fasciitis

Erythema nodosum

Experimental allergic encephalomyelitis

Evans syndrome

Fibromyalgia

Fibrosing alveolitis

Giant cell arteritis (temporal arteritis)

Giant cell myocarditis

Glomerulonephritis

Goodpasture syndrome

Granulomatosis with polyangiitis (GPA) (formerly called Wegener's granulomatosis)

Graves' disease

Guillain-Barré syndrome

Hashimoto's encephalitis

Hashimoto's thyroiditis

Hemolytic anemia

Henoch-Schönlein purpura

Gestational herpes

Hypogammaglobulinemia

Idiopathic Thrombocytopenic Purpura (ITP)

IgA nephropathy

IgG4-associated sclerosing disease

Immunoregulatory lipoproteins

Inclusion body myositis

Interstitial cystitis

Juvenile arthritis

Juvenile diabetes (Type 1 diabetes)

Juvenile myositis

Kawasaki syndrome

Lambert-Eaton syndrome

Leukocytoclastic vasculitis

Lichen planus

Lichen sclerosus

Woody conjunctivitis

Linear IgA disease (LAD or Linear IgA disease)

Lupus (SLE)

Lyme disease, chronic

Ménière's disease

Microscopic polyangiitis

Mixed connective tissue disease (MCTD)

Mooren ulcer

Mucha-Habermann disease

Multiple sclerosis

Myasthenia gravis

Myositis

Narcolepsy

Neuromyelitis optica (Devic)

Neutropenia

Ocular scar pemphigoid

Optic neuritis

Palindromic rheumatism

PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus)

Paraneoplastic cerebellar degeneration

Paroxysmal nocturnal hemoglobinuria (PNH)

Parry Romberg syndrome

Parsonnage-Turner syndrome

Pars planitis (peripheral uveitis)

Pemphigus

Peripheral neuropathy

Perivenous encephalomyelitis

Pernicious anemia

POEMS Syndrome

Polyarteritis nodosa

Autoimmune polyglandular syndromes Type I, II and III

Rheumatic polymyalgia

Polymyositis

Post-myocardial infarction syndrome

Postpericardiotomy syndrome

Progesterone dermatitis

Primary biliary cirrhosis

Primary sclerosing cholangitis

Psoriasis

Psoriatic arthritis

Idiopathic pulmonary fibrosis

Phagedenic pyoderma

Pure red blood cell aplasia

Raynaud's phenomenon

Reactive arthritis

Reflex sympathetic dystrophy

Reiter's syndrome

Recurrent polychondritis

Restless legs syndrome

Retroperitoneal fibrosis

Rheumatic fever

Rheumatoid arthritis

Sarcoidosis

Schmidt syndrome

Scleritis

Scleroderma

Sjogren's syndrome

Sperm and testicular autoimmunity

Stiff person syndrome

Subacute bacterial endocarditis (SBE)

Susac syndrome

Sympathetic ophthalmia

Takayasu arteritis

Temporal Arteritis/Giant Cell Arteritis

Thrombocytopenic purpura (TTP)

Tolosa-Hunt syndrome

Transverse myelitis

Type 1 Diabetes

Ulcerative colitis

Undifferentiated connective tissue disease (UCTD)

Uveitis

Vasculitis

Vesiculobullous dermatosis

Vitiligo

Wegener's granulomatosis (now called Granulomatosis with Polyangiitis or GPA).

INFLAMMATORY DISEASES TO TREAT OR PREVENT

Alzheimer's

ankylosing spondylitis

arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis)

asthma

atherosclerosis

Crohn's disease

colitis

dermatitis

diverticulitis

fibromyalgia

hepatitis

irritable bowel syndrome (IBS)

systemic lupus erythematosus (SLE)

nephritis

Parkinson disease

ulcerative colitis

The improved delivery possible using bacterial carrier cells, bacteriophages and phagemids as disclosed herein also has a wide range of non-medical applications, such as the treatment of bacteria associated with microbial corrosion or biological fouling of a substrate or fluid in an industrial or domestic system.

Consequently, the bacteriophage, phagemid and the support cells of the invention as disclosed herein may be in the form of a composition which is a medical, ophthalmic, dental or pharmaceutical composition. In one example, the composition is an antimicrobial composition, e.g. an antibiotic or antiviral, e.g. a medicine, disinfectant or mouthwash. In one example, the composition is a cosmetic composition (e.g. makeup composition for the face or body). In one example, the composition is a herbicide. In one example, the composition is a pesticide (e.g. when the target bacteria is Bacillus (e.g. thuringiensis)). In one example, the composition is a beverage additive (e.g., beer, wine or alcoholic beverage). In one example, the composition is a food additive (e.g. when the target bacteria is E coli, Salmonella, Listeria or Clostridium (e.g. botulinum)). In one example, the composition is a water additive. In one example, the composition is an additive for aquatic animal environments (e.g., in an aquarium). In one example, the composition is a composition from the petroleum or petrochemical industry or included in such a composition (e.g. when the target bacteria is a sulfate-reducing bacteria, e.g. a Desulfovibrio). In one example, the composition is a petroleum or petrochemical additive. In one example, the composition is a chemical additive. In one example, the composition is a disinfectant (e.g. for sterilizing equipment for human or animal use, e.g. for surgical or medical use, or for baby feeding). In one example, the composition is a personal hygiene composition for human or animal use. In one example, the composition is a composition for environmental use, e.g. for soil treatment or environmental decontamination (e.g. from wastewater, or from petroleum, a petrochemical or chemical product, e.g. when the target bacteria is a sulfate-reducing bacteria , e.g. a Desulfovibrio). In one example, the composition is a plant growth stimulator. In one example, the composition is a composition intended for use in petroleum, petrochemical, metal or mineral extraction. In one example, the composition is a fabric treatment or additive. In one example, the composition is a treatment or additive for skin, leather or suede. In one example, the composition is a dye/dye additive. In one example, the composition is a beverage (e.g. beer or wine) brewing or fermentation additive (e.g. when the target bacteria is a Lactobacillus). In one example, the composition is a paper additive. In one example, the composition is an ink additive. In one example, the composition is a glue additive. In one example, the composition is a human or animal or plant antiparasitic composition. In one example, the composition is an air additive (e.g., for air in or produced by air conditioning equipment, e.g., when the target bacteria is Legionella). In one example, the composition is an antifreeze additive (e.g. when the target bacteria is Legionella). In one example, the composition is an eye drop or an ophthalmic composition (e.g., contact lens fluid). In one example, the composition is comprised of a dairy food (e.g. the composition is in or is a milk or dairy product; the target bacteria being e.g. Lactobacillus, Streptococcus, Lactococcus or Listeria). In one example, the composition is or is comprised of a household or industrial cleaning product (e.g. when the target bacteria is E coli, Salmonella, Listeria or Clostridium (e.g. botulinum)). In one example, the composition is comprised of a fuel. In one example, the composition is comprised of a solvent (e.g. other than water). In one example, the composition is a cooking additive (e.g., a food cooking additive). In one example, the composition is a laboratory reagent (e.g., for use in biotechnology or recombinant DNA or RNA technology). In one example, the composition is comprised of a fiber retting agent. In one example, the composition is intended for use in a vitamin synthesis process. In one example, the composition is an anti-rot composition for crops or plants (e.g. when the target bacteria is a saprotrophic bacteria). In one example, the composition is an anti-corrosion compound, e.g. to prevent or reduce corrosion of metals (e.g. when the target bacteria is a sulfate-reducing bacteria, e.g. Desulfovibrio, e.g. for use in the reduction or prevention of corrosion of extraction, processing or containment equipment; metal extraction, processing or containment equipment; or mineral extraction, treatment or containment equipment). In one example, the composition is an agricultural or livestock composition or included in such a composition. In one example, the composition is a silage additive.

Any aspect of the present invention is intended for industrial or household use, or is used in a process for such use. For example, it is intended for or used in agriculture, the petroleum or hydrocarbon industry, the food or beverage industry, the clothing industry, the packaging industry, the electronics industry, computer industry, environmental industry, chemical industry, aerospace industry, automobile industry, biotechnology industry, medical industry, health industry, dental industry, dental industry energy, consumer products industry, pharmaceutical industry, mining industry, cleaning industry, forestry industry, fishing industry, leisure industry, manufacturing industry recycling, the cosmetics industry, the plastics industry, the pulp or paper industry, the textile industry, the clothing industry, the leather or suede or animal skins industry, the tobacco industry or the steel industry.

These different applications imply that the supporting bacterial cell comprising plasmids and or a conjugative bacteriophage(s) used is capable of providing MTase to bacteria associated with biofouling or microbial corrosion in these industries.

Indeed, it will be seen that the invention finds application in any process which generates an effect by the specific targeted modification (e.g. destruction) of a target bacterial cell in the context of another, so that the population relative of the targeted bacteria and another the bacteria is altered. This can have medical and non-medical applications.

All of these applications all also benefit from the combination of the use of MTases as disclosed herein in combination with components of the CRISPR system as described above, to modify the DNA of target bacteria, resulting for example in the destruction of target cells. .

It will be understood that the particular embodiments described herein are presented by way of illustration and not as limitations of the invention.

The main features of this invention can be used in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to determine using nothing more than routine investigation, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

All publications and patent applications referred to in the description are indicative of the level of skill of those skilled in the art to whom this invention relates. All publications and patent applications are incorporated herein by reference to the same extent as if each particular publication or patent application were specifically and individually stated to be incorporated by reference. The use of the word "a" or "an", when used in conjunction with the term "comprising" in the claims and/or description may mean "a", respectively "an", but it is also consistent with the meaning of “one or more”, “at least one” and “one or more than one”. The use of the term "or" in the claims is used to mean "and/or" unless it is explicitly stated to refer only to alternatives or the alternatives are mutually exclusive, although the disclosure supports a definition that refers only to alternatives and “and/or”.

Throughout this application, the term "approximately" is used to indicate that a value includes the variation in error inherent in the device, the method being employed to determine the value, or variation, which exists among the subjects of study.

As used in this specification and the claim(s), the words "comprising" (and any form of umbrella, such as "include" and "includes"), "have" (and any form of possession , such as “have” and “a”), “including” (and any form of inclusion, such as “includes” and “include”), or “containing” (and any form of containment, such as “contains” and “contain”) are inclusive or open and do not exclude the presence of additional elements or process steps not mentioned.

The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed elements preceding the term. For example, "A, B, C or combinations thereof" are intended to include at least one of the following: A, B, C, AB, AC, BC or ABC, and if the order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC or CAB. Continuing with this example, combinations that contain repetitions of one or more elements or terms, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so on, are expressly included. Those skilled in the art will understand that there is typically no limit to the number of elements or terms in any combination, unless the context indicates otherwise.

Any part of this disclosure may be read in conjunction with any other part of the disclosure, unless the context indicates otherwise.

Any compositions and/or methods disclosed and claimed herein may be prepared and carried out without undue experimentation in light of the present disclosure. Although the compositions and methods of this invention have been described using the preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the compositions and/or methods and to the steps or sequence of steps of the process mentioned here without departing from the concept, spirit and scope of the invention. All such substitutes and similar modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Examples

The present invention is described in more detail in the following non-limiting examples.

Processes and materials

Plasmids

All plasmids referred to in this document are described in Table A.

DNA fragments containing ORFs encoding the MTase activities of RM type I systems (M and S subunits) were amplified by PCR from appropriate E. coli strains. For the conjugation experiments, the DNA fragments coding for the type I MTases M.Eco0015 and M.EcoKI were cloned into the small conjugative plasmid p1114 which contains the mobilization and origin of the replication elements of pBBR1 (Antoine and Locht. Mol. Microbiol. (in English: “Microbiol. Mol.”) 6(13):1785-1799 (1992), or in the large conjugation plasmid pCol1B9SL1344 of the IncI1 incompatibility group (Kröger et al. PNAS . 109(20):E1277-E1286 (2012). (PNAS: "Proceedings of the National Academy of Sciences", or "Proceedings of the National Academy of Sciences") In both cases, the MTases are encoded on the DNA transferred to the recipient cells and thus the methylation of this DNA is said to be "in cis". For constructs based on p1114, a pair of each of the type I RM recognition sites separated by approximately 850 to 1350 bp was cloned to provide a substrate for the MR systems tested.

The MTase ORFs were also cloned into the non-conjugative plasmid p145 which has a compatible marker (tetracycline (Tet) resistance) and an origin of replication (pSC101) to co-reside in the same cells as the small conjugative plasmid p1114 ( devoid of any MTAse coding). Subsequent methylation of p1114 by the methylase activity encoded on a co-resident plasmid is said to be “in trans”. For each of the MTase-encoding plasmids, corresponding negative control plasmids were created in which stop codons were introduced into the orfs to suppress MTase activity without altering the remainder of the DNA sequence. Chloramphenicol (Cm) resistance was encoded on transferred DNA for a selection of transconjugants while expression from MTase ORFs was under the control of the arabinose promoter.

To methylate the DNA packaged into a P4 transducing phage particle, seven type I MTases (ORFs of the M and S subunits, which together provide the methylation activity) were cloned into p1114 to create the operon construct p1160. The order of MTases in the operon was EcoMII, EcoAI, EcoKI, EcoAO83I, EcoMIII, Eco0015 and Eco1167, with an arabinose promoter cloned 5' of the EcoMII sequence and 5' of the Eco0015 sequence. For packaging into P4 particles, plasmid p1065 was constructed to contain a P4 packaging signal, a spectinomycin resistance marker (Spec), the sGFP ORF under the control of the BBa_J23100 promoter (collection of Andersen promoters) and a pair of each of the type I recognition sites (as above) separated by approximately 850 to 1350 bp. The backbone of this plasmid is based on pCasens3 (Uribe et al. Cell Host & Microbe. 25:233-241 (2019)).

For expression of the Eco0015 RM system for use in lambda phage dot assays, plasmid p1331 was constructed by cloning a PCR fragment containing the hsdR-hsdM-hsdS ORFs of Eco0015, into plasmid p145. For weak constitutive expression, the pM0 promoter is located 5' of the ORF of the M subunit.

Bacterial strains and their culture

All strains were cultured in LB culture medium (from English: “lysogeny broth”) at 37°C with the addition of antibiotics, supplements or under culture conditions as indicated for the experiments.

All strains used for molecular biology procedures, including conjugation and preparation of phage lysates, are listed in Table B.

All strains used to test phage conjugation and lysis or transduction efficiency are listed in Table C.

For conjugation experiments, E. coli strain JKE201 (Harms et al. PLos Genet . 13(10): e1007077(2017)) was transformed with the conjugation plasmids and used as a donor. This strain contains the RP4 mating system on its chromosome and is produced auxotrophic for diaminopimelic acid (DAP), allowing counter-selection of transconjugants from donor cells.

To test the benefits of methylation on phage P4 conjugation and transduction, a panel of E. coli clinical isolates harboring type I RM systems, specifically EcoKI or Eco0015 RM systems, were selected as as recipient strains. A wild-type E. coli C-1 strain (strain CGSCG #: 3121), designated b52, was chosen as a comparison strain and does not have an active type I or type II restriction system.

The culturing efficiency (or eop, from English “efficiency of plating”) of lambda phages was tested by spot tests on several strains. To test eop against the EcoKI restriction system, strain MG1655 was used. To test eop against the Eco0015 system, strain NEB 10-beta was transformed with p1331 (harboring the hsdR-hsdM-hsdS system of Eco0015 under constitutive expression); and to test eop against EcoKI and Eco0015 in the same strain, MG1655 was transformed with p1331.

Strain b587 was the production strain for P4 packaged DNA.

Conjugation conditions

For conjugation experiments, cultures of donor strains were grown in LB culture medium supplemented with chloramphenicol (Cm 20 µg ml ^-1 ), DAP (100 µM) and arabinose (0.2% p /v). Overnight cultures of the donor strains were washed in Phosphate Buffered Saline (PBS) and resuspended in a volume of LB culture medium equal to that of the overnight culture while the recipient strains were grown overnight in LB culture medium without any supplement or culture used directly. In a 96-well microtiter plate, 50 μl of donor strain and 10 μl of recipient strain were mixed in 140 μl of LB culture medium, supplemented with DAP and arabinose. The conjugation reactions were left in stationary incubation at 37°C for 18 h. The transconjugant cultures were plated as 10-fold dilutions in PBS on LB agar plates supplemented with arabinose and Cm and incubated for 18 h at 37°C. The donor cells did not grow because they had required DAP, while transconjugant recipients, harboring the Cm gene on the donor plasmid, are capable of growth and were enumerated in cfu ml ^-1 of the transconjugant mixture.

Lambda phage engineering

Synthetic lambda phage genomes were designed to be assembled from PCR fragments using commercially available lambda DNA (NEB Cat# N3011S) as a template for PCR. The cI, rexA and rexB genes were excluded from the assembly designs and thus the subsequently restarted lambda phage is virulent. The Sam7 amber mutation (45.352 G->A), a mutation in a gene involved in cell lysis that is present in the commercial DNA frame, was reverted to wild-type sequence (A->G). Three genome designs were made, which, as described above without any additional sequences, are referred to as Design I (see Appendix I for genome sequence). Design II includes the hsdM and hsdS ORFs of EcoKI under the control of the Anderson pJ23100 promoter, amplified by PCR from p1172 and assembled in place of the deleted lambda genes (see Appendix II for the genomic sequence). Similarly, Design III includes the hsdM and hsdS ORFs of Eco0015, which were PCR amplified from p1174, but unlike Design II, their expression is under the control of the native lambda sequence and assembled instead deleted lambda genes (see Appendix III for the genomic sequence).

Platinum™ SuperFi™ High Fidelity DNA Polymerase (Invitrogen) was used in all PCR reactions. PCR primers were designed to incorporate BsaI sites to facilitate seamless Golden Gate assembly of PCR fragments. Assembled phage genomes were used to transform E. coli NEB 10-beta cells by electroporation. The transformation was mixed with 3 ml of LB top agar (0.4% w/v agar) containing 100 µl of overnight culture of NEB 10-beta and supplemented with 5 mM CaCl ₂ and 10 mM MgCl ₂ . After overnight incubation of the plates at 37°C, individual plates were isolated and propagated on relevant production strains to create lysates.

Lambda phage propagation and titration determination

For unmethylated lambda phage DNA, the phage was grown on NEB 10-beta, for trans methylation by MTase EcoKI phages were grown on MG1655, for trans methylation by MTase Eco0015 of phages were cultured on NEB-10 beta harboring the p1331 plasmid, for methylation in trans by the MTases EcoKI and Eco0015 phages were cultured on MG1655 harboring p1331, and for the production of lambda phages with genomic designs II and III (for cis methylation), the phage was propagated on NEB 10-beta. The strains were grown until the start of the exponential phase at 37°C in LB medium supplemented with 5 mM CaCl ₂ and 10 mM MgCl ₂ before the addition of phages. After overnight incubation, the cultures were centrifuged at 10,000 g for 5 min and the supernatant filtered through a 0.2 μm pore size filter. The titers of these resulting lysates were determined by titration on E. coli NEB 10-beta bacterial overlays. Overlays were created using 3 ml of LB top agar (0.4% agar) containing 100 µl of NEB 10-beta overnight culture and supplemented with 5 mM CaCl ₂ and 10 mM MgCl ₂ . The lysates were serially diluted 10 times in SM buffer (50 mM Tris-HCl (pH 7.5), 8 mM MgSO ₄ , 100 mM NaCl), placed on the bacterial overlay, and after incubation for one Overnight, the resulting plaques were counted to calculate an eop in pfu ml ^-1 of lysate.

Phage lambda spot test

All phage lysates were diluted in SM buffer (50 mM Tris-HCl (pH 7.5), 8 mM MgSO4, 100 mM NaCl) to an eop similar to that determined on E. coli NEB 10-beta. These were then serially diluted 10-fold in SM buffer and spotted onto bacterial overlays (performed as described above) of the relevant strains. For dot testing of cocktail lysates, equal volumes of the two lysates at identical titers were mixed before further dilution and dotting. To test the eop against the EcoKI restriction system, the E. coli MG1655 strain was used. To test the eop with regard to the Eco0015 system, the NEB 10-beta strain was transformed with p1331 (carrying the hsdR-hsdM-hsdS system of Eco0015 under constitutive expression). To test both the EcoKI and Eco0015 systems in the same strain, MG1655 was transformed with p1331.

Preparation and titration of P4 transducing phage lysate

Plasmid p1065 (containing sGFP, a spectinomycin marker, type I restriction recognition sites and a COS P4 packaging site as described above) was used to transform the E. coli strain. coli b587 which contains all the genetic machinery necessary to catalyze the packaging of p1065 into phage heads. For methylation of packaged DNA, plasmid p1160 (containing an MTase operon under the control of the arabinose promoter) was cotransformed into b587.

Overnight cultures of the transformants were grown in LB medium (supplemented with Tet + Spec + 5 mM CaCl ₂ and 10 mM MgCl ₂ ) and incubated at 37°C, with shaking. The next day, fresh 10 ml cultures were inoculated at OD ₆₀₀ =0.1 then grown to OD ₆₀₀ =0.2, at which time the packaging and lysis genes, and in the case of p1160 the expression of MTases, were induced by the addition of arabinose (0.2% w/v). The cultures were left to lyse for 3-4 h at 37°C with shaking. The lysed cultures were centrifuged (5 minutes at 10,000xg) and the supernatant filter was sterilized using a 2uM filter. To remove residual bacterial DNA, the lysate was incubated with DNaseI overnight at 37°C.

To determine the lysate titer, 100 μl of lysate was mixed with 100 μl of overnight culture of strain b52 and incubated at room temperature for 30 minutes. This mixture was serially diluted 1:10 in SM buffer, spotted onto LB+Spec plates and GFP positive colonies were counted to calculate a titer in cfu ml ^-1 of lysate. The titer on clinical strains was determined by the same method.

The results of the experiments are shown in Figures 1 to 5

. Conjugation efficiency of small cis-methylated conjugative plasmids (n=3)

A. Small conjugative plasmids encoding wild-type (p1172 and p1174) or mutated (p1243 and p1244) versions of M.EcoKI and M.Eco0015 are directly compared for conjugation efficiency in clinical isolates of E.coli . Strain b52 is used as a negative control because it does not harbor any active restriction system. Bars marked with an X are for strains harboring the EcoKI restriction system while all other strains harbor the Eco0015 restriction system. The dashed line represents the detection limit at 2.3 log.

B. Unpaired T-test comparison of the conjugation efficiency of p1174 (encoding wild-type M.Eco0015) and p1244 (encoding mutated M.Eco0015) in strains harboring the Eco0015 restriction system (p < 0 .05).

. Conjugation efficiency of a large cis-methylated conjugative plasmid (n = 3)

A. Large conjugative plasmids based on pCol1B9SL1344 and encoding wild-type (p1284) or mutated (p1286) versions of M.Eco0015 respectively are directly compared for conjugation efficiency in strains from clinical collections of E. coli .

The b52 strain is used as a negative control because it does not harbor any active restriction system. Bars marked with an X are for strains harboring the EcoKI restriction system while all other strains harbor the Eco0015 restriction system.

The dashed line represents the detection limit at 2.3 log.

B. Unpaired T-test comparison of the conjugation efficiency of p1284 (encoding wild-type M.Eco0015) and p1286 (encoding mutated M.Eco0015) in strains harboring the Eco0015 restriction system (p < 0 .05).

. Comparison of the conjugation efficiency of small conjugative plasmids methylated in cis and trans (n=3)

A. The small conjugative plasmid (p1114) is methylated in trans by a non-conjugative coresident plasmid encoding respectively either wild type (p1082 and p819) or mutated type (p1369 and p1368) versions of M.EcoKI and M.Eco0015 (n=3). Conjugation efficiency of trans-methylated plasmids is plotted against cis-methylation conjugation efficiency data of the (A), with plasmids encoding wild-type (p1172 and p1174) and mutated (p1243 and p1244) versions of M.EcoKI and M.Eco0015 respectively. Strain b52 is used as a negative control because it does not harbor any active restriction system. Bars marked with an X are for strains harboring the EcoKI restriction system while all other strains harbor the Eco0015 restriction system. The dotted line represents the detection limit of the assay at 2.3 logs.

B. Unpaired T-test comparison of the conjugation efficiency of p1082 (encoding M.Eco0015 in wild-type trans) and p1174 (encoding M.Eco0015 in wild-type cis) in strains harboring the Eco0015 restriction (p < 0.05).

. Efficiency of culturing phage lysates, methylated either in trans or in cis (n=3)

On the horizontal axis is the background description of the strain in which the phage lysates were made with respect to methylation properties. The categories being: unmethylated phages, trans-methylated phages e.g. from MTases encoded by the genome of the strain or on a co-resident plasmid, and methylation in cis e.g. from MTases encoded on the phage genome. In some cases, two lysates were produced separately and then mixed to form a cocktail. Each graph shows the eop of identification of these lysates on a strain of E. coli with particular restriction properties:

A. eop on a strain containing no restriction enzyme (NEB 10-beta)

B. eop on strain harboring the EcoKI system (MG1655)

C. eop on strain harboring the Eco0015 system (NEB 10-beta containing p1331)

D. eop on a strain harboring both EcoKI and Eco0015 systems (MG1655 containing p1331)

. Efficiency of phage transduction after methylation in trans by a production strain expressing an operon MTase (n=3)

Methylated and unmethylated p1065 plasmid was packaged into a P4 transducing phage particle and transduction efficiency was compared in a panel of E. coli clinical isolates. coli. DNA methylation of p1065 was carried out in trans by producing a lysate from a strain harboring a coresident plasmid (p1160) encoding an operon of seven MTases. The b52 strain is used as a negative control because it does not harbor any active restriction system. Strains marked with an p1160 but show no improvement in transduction efficiency. All other strains do not encode a restriction system related to the MTases encoded by p1160. The dotted line represents the detection limit of the assay at 2.3 logs.

Details of various MTases used in these experiments are provided in Appendix IV.

Appendix V is the sequence of p1065

Conclusions

We can see that :

Conjugative plasmids encoding MTase enzymes are significantly protected from host RM systems after conjugation;

Protection may be offered by methylation in cis or trans (e.g. by a non-conjugative plasmid co-resident in the mating strain);

Cocktails of MTases can provide high levels of protection against MR systems;

and operons encoding multiple MTases can be used to efficiently methylate plasmid DNA for delivery by phage transduction.

Table A: Plasmids Plasmid ID Description Insert key features* Marker pen OriR Skeleton Source p1114 Conjugation Cloning Skeleton mobilizable elements of pBBR1, SpisPink gene in the cloning site cm pBBR1 rep The backbone sequence is derived from pBBR1-mcs2 and is identical to GeneBank entry MH539767 p145 Cloning vector SpisPink gene in the cloning site Tet pSC101 The backbone sequence is derived from pLG388 and is identical to GenBank entry KM604642 p1172 Wild-type MTase EcoK1 in small conjugative plasmid AraCpBAD-EcoK1 M+S orfs, 2x copies of each Type I restriction site cm pBBR1 rep P1114 p1174 Wild-type MTase Eco0015 in small conjugative plasmid Orfs AraCpBAD-Eco0015 M+S, 2x copies of each Type I restriction site cm pBBR1 rep P1114 p1243 Mutant-like MTase EcoK1 in small conjugative plasmid Orfs AraCpBAD-EcoK1 M (stop codon) + S (stop codon), 2x copies of each Type I restriction site cm pBBR1 rep P1114 p1244 MTase Eco0015 mutant type in small conjugative plasmid Orfs AraCpBAD-Eco0015 M (stop codon) + S (stop codon), 2x copies of each Type I restriction site cm pBBR1 rep P1114 p1284 Wild-type MTase Eco0015 in large conjugative plasmid Orfs AraCpBAD-Eco0015 M+S cm repA The backbone sequence is derived from pCol1B9SL1344 and is identical to GenBank entry HE654725 p1286 Mutant-like MTase Eco0015 in large conjugative plasmid Orf AraCpBAD-Eco0015 M (stop codon) + S (stop codon) cm repA The backbone sequence is derived from pCol1B9SL1344 and is identical to GenBank entry HE654725 p819 Wild-type MTase EcoK1 in cloning backbone AraCpBAD-EcoK1 M+S orfs, 2x copies of each Type I restriction site Tet pSC101 p145 p1082 Wild-type MTase Eco0015 in cloning backbone Orfs AraCpBAD-Eco0015 M+S, 2x copies of each Type I restriction site Tet pSC101 p145 p1368 Mutant-like MTase EcoK1 in cloning backbone Orfs AraCpBAD-EcoK1 M (stop codon) + S (stop codon), 2x copies of each Type I restriction site Tet pSC101 p145 p1369 Mutant-like MTase Eco0015 in cloning backbone Orfs AraCpBAD-Eco0015 M (stop codon) + S (stop codon), 2x copies of each Type I restriction site Tet pSC101 p145 p1331 Eco0015 restriction system in cloning skeleton Orfs pM0-Eco0015 R + M + S Tet pSC101 p145 p1160 Type I MTase operon in small conjugative plasmid AraCpBAD-EcoMII-EcoAI-EcoKI-EcoAO83I-EcoMIII-pBAD-Eco0015-Eco1167 (M + S orfs of each) cm pBBR1 rep P1114 p1065** Plasmid for packaging into P4 particles pJ23100-sGFP, AraCpBAD-delta(P2 activation)-psu, P4 packaging signal Spec CloDF13 (mutation gives approx. 70 copies/cell) Based on pCasens3, see Appendix V for DNA sequence

Table B: General molecular biology of E. coli Strain ID Description Key notes b3235 JKE201 donor strain, DAP auxotroph, encodes the RP4 type IV secretion system b230 MG1655 strain K-12, code for the EcoKI type I RM system b2522 NEB 10-beta strain K-12, derived from DH10B, no active RM system b587 Strain C-1a P2 wt lysogen, P2 receptor deletion, zeocin resistant

Table C: Strains of E. coli to test the efficiency of phage conjugation and transduction Strain ID Description GMO Type I and II MR systems* B-52 Strain C-1a 1 - b292 ECOR33 1 - b294 ECOR35 2 - b315 ECOR56 2 Eco31I (type II), EcoMII (type I) b325 ECOR66 2 - b331 ECOR72 2 - b662 ESBL20170384 2 EcoMIII (type I), Eco89II (type I) b682 ESBL20180320 2 Eco0008 (type I), Eco736 (type II) b692 ESBL20150072 2 Eco1167 (type I) b751 SEATTLE_BLOOD-08-0654 2 Eco0015 (type I) b755 SEATTLE_BLOOD-10-0686 2 Eco0015 (type I) b762 SEATTLE_BLOOD-10-0687 2 Eco0015 (type I) b766 SEATTLE_BLOOD-09-0751 2 Eco0015 (type I) b770 SEATTLE_BLOOD-08-0493 2 Eco0015 (type I) b771 SEATTLE_BLOOD-09-1294 2 EcoKI (type I), Eco1246 (type I) b803 SEATTLE_UPEC-76 2 Eco0015 (type I) b819 SEATTLE_UPEC-129 2 Eco0015 (type I) b821 SEATTLE_UPEC-169 2 Eco0015 (type I) b830 SEATTLE_UPEC-186 2 EcoAO83I (type I) b838 SEATTLE_UPEC-124 2 Eco31I (type II) b848 SEATTLE_UPEC-142 2 Eco31I (type II), EcoMII (type I), EcoMIII (type I) b854 SEATTLE_UPEC-120 2 Eco0015 (type I) b867 SEATTLE_UPEC-211 2 Eco0015 (type I), Eco1167 (type I) b879 SEATTLE_UPEC-256 2 EcoKI (type I)

*RM system designations taken from known REBASE recognition sequences

Table D: Example of phage Tevenvirinae

Acinetobacter virus 133

Aeromonas 65 virus

Aeromonas virus Aeh1

Dhakavirus

Escherichia virus Bp7

Escherichia virus IME08

Escherichia virus JS10

Escherichia virus JS98

Escherichia virus MX01

Escherichia virus QL01

Escherichia virus VR5

Escherichia virus WG01

Escherichia Phage RB16

Escherichia Phage RB32

Escherichia virus RB43

Enterobacteria phage RB43-GVA

Gaprivervirus

Escherichia virus VR20

Escherichia virus VR25

Escherichia virus VR26

Escherichia virus VR7

Shigella virus SP18

Gelderlandvirus

Salmonella Melville Phage

Salmonella S16 Phage

Salmonella Phage STML198

Salmonella phage STP4a

Jiaodavirus

Klebsiella virus JD18

Klebsiella virus PKO111

Karamvirus

Enterobacter PG7 virus

Escherichia virus CC31

Krischvirus

Escherichia virus ECD7

Escherichia virus GEC3S

Escherichia virus JSE

Escherichia phi1 virus

Escherichia virus RB49

Moonvirus

Citrobacter CF1 virus

Citrobacter Merlin virus

Citrobacter Moon Virus

Mosigviruses

Escherichia virus APCEc01

Escherichia HP3 virus

Escherichia virus HX01

Escherichia virus JS09

Escherichia virus O157tp3

Escherichia virus O157tp6

Escherichia virus PhAPEC2

Escherichia virus RB69

Escherichia virus ST0

Shigella virus SHSML521

Shigella virus UTAM

Schizotequatrovirus

Vibrio KVP40 virus

Vibrio nt1 virus

Vibrio ValKK3 virus

Slopekvirus

Enterobacter Eap3 virus

Klebsiella virus KP15

Klebsiella virus KP27

Klebsiella Matisse virus

Klebsiella Miro virus

Klebsiella virus PMBT1

Tequatrovirus

Enterobacteria T4 phage sensu lato

Escherichia virus AR1

Escherichia virus C40

Escherichia CF2 virus

Escherichia virus E112

Escherichia virus ECML134

Escherichia virus HY01

Escherichia virus HY03

Escherichia virus Ime09

Escherichia virus RB14

Escherichia RB3 virus

Escherichia virus slur03

Escherichia virus slur04

Escherichia virus SV14

Escherichia virus T4

Shigella virus Pss1

Shigella virus Sf21

Shigella virus Sf22

Shigella virus Sf24

Shigella virus SHBML501

Shigella virus Shfl2

Vibrio nt-1 phage sensu lato

Yersinia virus D1

Yersinia virus PST

Unclassified Tevenvirinae

Acinetobacter phage AbTZA1

Acinetobacter phage Ac42

Acinetobacter phage Acj61

Acinetobacter Phage Acj9

Acinetobacter phage AM101

Acinetobacter Phage Henu6

Acinetobacter phage KARL-1

Acinetobacter phage vB_AbaM_PhT2

Acinetobacter phage vB_AbaP_Abraxas

Acinetobacter phage vB_ApiM_fHyAci03

Acinetobacter ZZ1 phage

Aeromonas Phage 65.2

Aeromonas Phage Ah1

Aeromonas Phage AS-sw

Aeromonas Phage AS-szw

Aeromonas Phage AS-yj

Aeromonas Phage AS-zj

Aeromonas Phage AsFcp_1

Aeromonas Phage AsFcp_2

Aeromonas Phage AsFcp_4

Aeromonas Phage Assk

Aeromonas Phage Asswx_1

Aeromonas Phage AsSzw2

Aeromonas Phage Aswh_1

Aeromonas Phage Aszh-1

Aeromonas Phage CC2

Aeromonas phiAS5 Phage

Aeromonas Phage PX29

Buttiauxella phage vb_ButM_GuL6

Citrobacter phage IME-CF2

Citrobacter Margaery Phage

Citrobacter Maroon Phage

Citrobacter Miller Phage

Citrobacter phage vB_CfrM_CfP1

Cronobacter S13 phage

Cronobacter phage vB_CsaM_GAP161

Cronobacter phage vB_CsaM_leB

Cronobacter phage vB_CsaM_leE

Cronobacter phage vB_CsaM_leN

Edwardsiella Phage PEi20

Edwardsiella Phage PEi26

Enterobacter phage EBPL

Enterobacter EC-F1 phage

Enterobacter EC-F2 phage

Enterobacter EC-W1 phage

Enterobacter EC-W2 phage

Enterobacter phage vB_EclM_CIP9

Erwinia Cronus Phage

Escherichia Phage Lw1

Escherichia Phage RDN37

Klebsiella phage AmPh_EK29

Klebsiella EI phage

Klebsiella phage KOX11

Klebsiella phage KOX8

Klebsiella phage KPN1

Klebsiella Marfa Phage

Klebsiella phage PhiKpNIH-6

Klebsiella phage vB_Kpn_F48

Klebsiella phage vB_Kpn_P545

Klebsiella phage vB_KpnM_Potts1

Morganella phage vB_MmoM_MP1

Panteoa Phynn Phage

Pectobacterium PM2 bacteriophage

Proteus phage phiP4-3

Proteus Phage PM2

Proteus phage vB_PmiM_Pm5461

Pseudomonas Phage PspYZU05

Serratia Muldoon Phage

Phage of Serratia PS2

Shewanella Thanatos-1 Phage

Shewanella Thanatos-2 Phage

Shigella phage vB_SdyM_006

Sinorhizobium phage vB_SmelM_phiM10

Sinorhizobium phage vB_SmelM_phiM14

Vibrio phage vB_VmeM-32

Yersinia Phage JC221

TABLE E: Medications ACE (Angiotensin-Converting Enzyme or ACE) inhibitors with calcium channel blocking agents
ACE inhibitors with thiazides
adamantane antivirals
adrenocortical steroids
adrenal corticosteroid inhibitors
adrenergic bronchodilators
agents for hypertensive emergencies
agents for pulmonary hypertension
aldosterone receptor antagonists
alkylating agents
allergens
alpha-glucosidase inhibitors
alternative medications
amoebicides
aminoglycosides
aminopenicillins
aminosalicylates
AMPA receptor antagonists
amylin analogues
analgesic combinations
painkillers
androgenic and anabolic steroids
angiotensin converting enzyme inhibitors
angiotensin II inhibitors with calcium channel blockers
angiotensin II inhibitors with thiazides
angiotensin receptor blockers
angiotensin receptor blockers and neprilysin inhibitors
anorectal preparations
anorectics
antacids
anthelmintics
anti-angiogenic ophthalmic agents
anti-CTLA-4 monoclonal antibodies
anti-infectious
anti-PD-1 monoclonal antibodies
antiadrenergic agents (central) with thiazides
antiadrenergic agents (peripheral) with thiazides
antiadrenergic agents, acting centrally
antiadrenergic agents, acting peripherally
antiandrogens
antianginal agents
antiarrhythmic agents
anti-asthma associations
antibiotics/antineoplastics
anticholinergic antiemetics
anticholinergic antiparkinsonian agents
anticholinergic bronchodilators
anticholinergic chronotropic agents
anticholinergics/antispasmodics
anticoagulant reversal agents
anti coagulants
anticonvulsants
antidepressants
antidiabetic agents
antidiabetic associations
antidiarrheals
antidiuretic hormones
antidotes
antiemetic/antivertigo agents
antifungals
antigonadotropic agents
anti-gout agents
antihistamines
antihyperlipidemic agents
antihyperlipidemic associations
antihypertensive combinations
antihyperuricemic agents
antimalarial agents
antimalarial associations
antimalarial quinolines
antimetabolites
antimigraine agents
antineoplastic detoxifying agents
antineoplastic interferons
antineoplastics
antiparkinsonian agents
antiplatelet agents
antipseudomonas penicillins
antipsoriatics
antipsychotics
antirheumatic drugs
antiseptics and germicides
antithyroid agents
antitoxins and antivenoms
anti-tuberculosis agents
anti-tuberculosis associations
cough suppressants
antiviral agents
antiviral boosters
antiviral associations
antiviral interferons
anxiolytics, sedatives and hypnotics
aromatase inhibitors
atypical antipsychotics
azole antifungals
bacterial vaccines
barbiturate anticonvulsants
barbiturates
BCR-ABL tyrosine kinase inhibitors
benzodiazepine anticonvulsants
benzodiazepines
beta blockers with calcium channel blockers
beta blockers with thiazides
beta-adrenergic blocking agents
beta-lactamase inhibitors
bile acid sequestrants
Organic Products
bisphosphonates
bone morphogenetic proteins
bone resorption inhibitors
bronchodilatory associations
bronchodilators
calcimimetics
calcineurin inhibitors
calcitonin
calcium channel blocking agents
anticonvulsants with carbamate(s)
carbapenems
carbonic anhydrase inhibitors decongestants
dermatological agents
diagnostic radiopharmaceuticals
diarylquinolines
dibenzazepine anticonvulsants
digestive enzymes
dipeptidyl peptidase 4 inhibitors
diuretics
dopaminergic antiparkinsonian agents
drugs used in alcohol dependence
echinocandins
EGFR inhibitors
estrogen receptor antagonists
estrogen
expectorants
factor Xa inhibitors
anticonvulsants derived from fatty acids
fibric acid derivatives
first generation cephalosporins
fourth generation cephalosporins
agents of functional intestinal disorders
gallstone solubilizing agents
gamma-aminobutyric acid analogues
gamma-aminobutyric acid reuptake inhibitors
gastrointestinal agents
general anesthetics
agents of the genitourinary tract
gastrointestinal stimulants
glucocorticoids
glucose level raising agents
glycopeptide antibiotics
glycoprotein platelet inhibitors
glycylcyclines
gonadotropin-releasing hormones
gonadotropin-releasing hormone antagonists
gonadotropins
group I antiarrhythmics
group II antiarrhythmics
group III antiarrhythmics
group IV antiarrhythmics
group V antiarrhythmics
growth hormone receptor blockers
growth hormones
guanylate cyclase-C agonists
H. pylori eradication agents
H2 antagonists
hedgehog pathway inhibitors
hematopoietic stem cell mobilizer
heparin antagonists
heparins
HER2 inhibitors
plant products
histone deacetylase inhibitors
hormones
hormones/antineoplastics
hydantoin anticonvulsants
hydrazide derivatives
immunoglobulins
immunological agents
immunostimulants
immunosuppressive agents
agents against impotence
in vivo diagnostic biological products
incretin mimetics
inhaled anti-infectives
inhaled corticosteroids
inotropic agents
insulin
insulin-like growth factor
integrase strand transfer inhibitor
let's interfere
interleukin inhibitors
interleukins
intravenous nutritional products
iodized contrast media
ionic iodinated contrast media
ferrous products
ketolides
laxatives
leprostatics
leukotriene modifiers
lincomycin derivatives
local injectable anesthetics
local injectable anesthetics with corticosteroids
loop diuretics
pulmonary surfactants
lymphatic coloring agents
lysosomal enzymes
macrolide derivatives
macrolides
contrast agents for magnetic resonance imaging
mast cell stabilizers
medical gases
meglitinides
metabolic agents
methylxanthines
mineralocorticoids
minerals and electrolytes
agents
painkillers
antibiotics
anticonvulsants
antidepressants
antidiabetic agents
antiemetics
antifungals
antihyperlipidemic agents
antihypertensive combinations
antimalarials
antineoplastics
antiparkinsonian agents
antipsychotic agents
anti-tuberculosis agents
antivirals
anxiolytics, sedatives and hypnotics
bone resorption inhibitors
cardiovascular agents
central nervous system agents
coagulation modifiers
diagnostic dyes/tinctures
diuretics
agents of the genitourinary tract
gastrointestinal agents (from the English “GI agents”)
hormones
metabolic agents
ophthalmic agents respiratory agents
sex hormones
topical agents
uncategorized agents
vaginal agents
mitotic inhibitors
monoamine oxidase inhibitors
mouth and throat products
mTOR inhibitors
mucolytics
multikinase inhibitors
muscle relaxers
mydriatics
narcotic analgesic combinations
narcotic painkillers
nasal anti-infectives
antihistamines and nasal decongestants
nasal lubricants and irrigations
nasal preparations
nasal steroids
natural penicillins
neprilysin inhibitors
neuraminidase inhibitors
neuromuscular blocking agents
neuronal potassium channel openers
next generation cephalosporins
nicotinic acid derivatives
NK1 receptor antagonists
Non-nucleoside reverse transcriptase inhibitors (NNRTIs)
non-cardioselective beta-blockers
non-iodized contrast media
non-ionic iodinated contrast media
non-sulfonylureas
nonsteroidal anti-inflammatory agents
NS5A inhibitors
Nucleoside Reverse Transcriptase Inhibitors (NRTIs)
nutraceutical products
nutritional products
ophthalmic anesthetics
ophthalmic anti-infectives
ophthalmic anti-inflammatory agents
antihistamines and ophthalmic decongestants
ophthalmic diagnostic agents
ophthalmic glaucoma treatment agents
ophthalmic lubricants and irrigations
ophthalmic preparations
ophthalmic steroids
ophthalmic steroids with anti-infectives
ophthalmic surgical agents
oral dietary supplements
immunostimulants
immunosuppressants
otic anesthetics
otic anti-infectives
otic preparations
otic steroids
otic steroids with anti-infectives
oxazolidinedione anticonvulsants
oxazolidinone antibiotics
parathyroid hormone and analogues
PARP inhibitors
PCSK9 inhibitors
penicillinase-resistant penicillins
penicillins
peripheral opioid receptor antagonists
mixed peripheral opioid receptor agonists/antagonists
peripheral vasodilators
peripherally acting antiobesity agents
phenothiazine antiemetics
phenothiazine antipsychotics
phenylpiperazine antidepressants
phosphate binders
plasma expanders
platelet aggregation inhibitors
platelet stimulating agents
polyenes
potassium-sparing diuretics with thiazides
potassium-sparing diuretics
probiotics
progesterone receptor modulators
progestins
prolactin inhibitors
prostaglandin D2 antagonists
protease inhibitors
protease-activated receptor 1 antagonists
proteasome inhibitors
proton pump inhibitors
psoralens
psychotherapeutic agents
psychotherapeutic associations
purine nucleosides
pyrrolidine anticonvulsants
quinolones
radiocontrast agents
radiological aids
radiological agents
radiological conjugating agents
radiopharmaceuticals
recombinant human erythropoietins

anticonvulsants
carbonic anhydrase inhibitors
cardiac stressors
cardioselective beta blockers
cardiovascular agents
catecholamines
CD20 monoclonal antibodies
CD30 monoclonal antibodies
CD33 monoclonal antibodies
CD38 monoclonal antibodies
CD52 monoclonal antibodies
central nervous system agents
cephalosporins
beta-lactamase inhibitors/cephalosporins
cerumenolytics
CFTR associations
CFTR potentiators
chelating agents
chemokine receptor antagonists
otic agents
renin inhibitors
respiratory agents
inhaled respiratory products
rifamycin derivatives
salicylates
sclerosing agents
second generation cephalosporins
selective estrogen receptor modulators
selective immunosuppressants
selective phosphodiesterase-4 inhibitors
selective serotonin reuptake inhibitors
serotonin-norepinephrine reuptake inhibitors
serotonergic neuroenteric modulators
sex hormone associations
sex hormones
SGLT-2 inhibitors
skeletal muscle relaxing associations
skeletal muscle relaxants
smoking cessation agents
somatostatin and somatostatin analogues
spermicides
statins
sterile irrigation solutions
streptomyces derivatives
succinimide anticonvulsants
sulfonamides
sulfonylureas
synthetic ovulation stimulants
tetracyclic antidepressants
tetracyclines
therapeutic radiopharmaceuticals
therapeutic vaccines
thiazide diuretics
thiazolidinediones
thioxanthenes
third generation cephalosporins
thrombin inhibitors
thrombolytics
thyroid medications
TNF alpha inhibitors
tocolytic agents
topical acne agents
topical agents
topical anesthetics
topical anti-infectives
topical anti-rosacea agents
topical antibiotics
topical antifungals
topical antihistamines
topical antineoplastics
topical antipsoriatics
topical antivirals
topical antivirals
topical debridement agents
topical depigmenting agents
topical emollients
topical keratolytics
topical nonsteroidal anti-inflammatories
topical photochemotherapy
topical rubefacient
topical steroids
topical steroids with anti-infectives
triazine anticonvulsants
tricyclic antidepressants
trifunctional monoclonal antibodies
ultrasound contrast agents
upper respiratory associations
urea anticonvulsants
agents of urea cycle disorders
urinary anti-infectives
urinary antispasmodics
urinary pH modifiers
uterotonic agents
vaccine combinations
vaginal anti-infectives
vaginal preparations
vasodilators
vasopressin antagonists
vasopressors
VE.GF/VE.G.FR inhibitors
viral vaccines
viscosupplementation agents
combinations of vitamins and minerals
vitamins
5-alpha-reductase inhibitors
5-aminosalicylates
5HT3 receptor antagonists
chloride channel activators
cholesterol absorption inhibitors
cholinergic agonists
cholinergic muscle stimulants
cholinesterase inhibitors
CNS stimulants
coagulation modifiers
colony stimulating factors
contraceptives
corticotropin
coumarins and indandiones
cox-2 inhibitors

Appendix I: Lambda Conception I phage restart sequence (virulent lambda) (SEQ ID NO: 2)

(sequence begins at COS site)

These sequences are those mentioned in the methods section under "Lambda Phage Engineering" and are used in the experiment for the .

Appendix II: Lambda Conception II phage restart sequence (virulent lambda, MTase EcoKI ) (SEQ ID NO: 3)

(sequence begins at COS site)

Appendix III: Lambda Conception II phage restart sequence (virulent lambda, MTase Eco0015) (SEQ ID NO: 4)

(sequence begins at COS site)

Annex IV Files of DNA sequences GenBank of orfs encoding RM subunits

LOCUS M.EcoKI 2981 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 5)

DEFINITION .

FEATURES Location/Qualifiers

misc_feature 1..1590

/label="M"

CDS 1..1590

/label="Translation 1-1590"

misc_feature 1587..2981

/label="S"

CDS 1587..2981

/label="Translation 1587-2981"

ORIGIN

1 atgaacaata acgatctggt cgcgaagctg tggaagctgt gcgacaacct gcgcgatggc

61 ggcgtttcct atcaaaacta cgtcaatgaa ctcgcctcgc tgctgttttt gaaaatgtgt

121 aaagagacgg gtcaggaagc ggaatacctg cggaaggtt accgctggga tgacctgaaa

181 tcccgcatcg gccaggagca gttgcagttc taccgaaaaa tgctcgtgca tttaggcgaa

241 gatgacaaaa agctggtaca ggcagttttt cataatgtta gtaccaccat caccgagccg

301 aaacaaataa ccgcactggt cagcaatatg gattcgctgg actggtacaa cggcgcgcac

361 ggtaagtcgc gcgatgactt cggcgatatg tacgaagggc tgttgcagaa gaacgcgaat

421 gaaaccaagt ctggtgcagg ccagtacttc accccgcgtc cgctgattaa aaccattatt

481 catctgctga aaccgcagcc gcgtgaagtg gtgcaggacc cggcggcagg tacggcgggc

541 tttttgattg aagccgaccg ctatgttaag tcgcaaacca atgatctgga cgaccttgat

601 ggcgacacgc aggatttcca gatccaccgc gcgtttatcg gcctcgaact ggtgcccggc

661 acccgtcgtc tggcactgat gaactgcctg ctgcacgata ttgaaggcaa cctcgaccac

721 ggcggcgcaa tccgtctggg caacacTctg ggTagcgacg gtgaaaacct gccgaaggcg

781 catattgtcg ccactaaccc gccgtttggc agcgccgcag gcaccaacat tacccgcacc

841 tttgttcacc cgaccagcaa caaacagTtg tgctttatgc agcatattat cgaaacgctg

901 catcccggcg gtcgtgcggc ggtggtggtg ccGgataacg tgCtgtttga aggcggcaaa

961 ggcaccgaca tTcgtcgtga cctgatggat aagtgtcatc tgcacaccat tctgcgtctg

1021 ccgaccggta ttttttacgc Tcagggcgtg aagaccaacg tgctgttctt taccaaaggG

1081 acGgtggcga acccgAatca ggataagaac tgTaccgaTg aTgtgtgggt gtatgacctg

1141 cgtaccaata tgccgagttt cggcaagcgc acaccgttta ccgacgagca tttgcagccg

1201 tttgagcgcg tgtatggcga agacccgcac ggtttaagcc cgcgcactga aggtgaatgg

1261 agttttaacg ccgaagagac ggaagttgcc gacagcgaag agaacaaaaa caccgaccag

1321 catcttgcta ccagccgctg gcgcaagttc agccgtgagt ggatccgcac cgcaaaatcc

1381 gattcgctgg atatctcctg gctgaaagat aaagacagta ttgatgccga cagcctgccg

1441 gagccggatg tattagcggc agaagcgatg ggcgaactgg tacaggcgct gtctgaactg

1501 gatgcgctga tgcgtgaact gggggcgagc gatgaggccg atttgcagcg tcagttgctg

1561 gaagaagcgt ttggtggggt gaaggaatga gtgcggggaa attgccggag gggtgggtta

1621 tcgccccagt atctacggtc acaactctaa tccgaggagt aacgtataaa aaagagcagg

1681 caataaatta tctaaaagat gattatttgc ctcttatccg tgcgaacaat attcagaatg

1741 gcaagtttga tactacggac ttggtttttg ttcctaaaaa tcttgttaaa gaaaGtcaaa

1801 aaatatctcc tgaagatatt gttattgcaa tgtcatcagg gagcaaatcc gtagttggta

1861 aatccgcaca tcagcatcta ccatttgaat gtagtttcgg cgcattttgc ggtgtattac

1921 gtcctgaaaa acttatattt tctggtttta ttgctcattt cacaaaatct tctctttatc

1981 gaaacaaaat ttcatcactt tctgctggtg caaatattaa taatattaag ccggcaagct

2041 ttgatttgat aaatatacca atcccaccac ttgccgaaca aaaaatcatc gctgaaaaac

2101 tcgatacgct gctggcgcag gtagacagca ccaaagcacg ttttgagcaa atcccacaaa

2161 tcctgaaacg ttttcgtcaa gcggtattgg ggggcgcagt taatggaaaa ttgacagaaa

2221 aatggcgtaa ttttgagccg caacattctg tatttaagaa gttaaatttt gaatctatct

2281 taactgaatt acgtaatggt ctttcatcaa agccaaatga aagtggtgtt ggtcatccaa

2341 tactacgcat tagttctgta cgtgctggcc atgtagatca aaacgatatt cggtttctag

2401 aatgttcaga aagtgaacta aaccgccaca aattacaaga tggagatctt ttatttactc

2461 gctataacgg aagtttagaa tttgttggtg tttgtgggtt attgaaaaaa ttacaacatc

2521 aaaatttgct atatcctgat aaacttattc gagctcgatt aaccaaagat gctttaaccag

2581 aatatatcga aatatttttt tcatccccct cagcacgaaa tgcaatgatg aactgcgtga

2641 aaacaacttc tggtcaaaaa ggtatttcag gaaaagatat caaatcccaa gttgttttat

2701 tacctccagt aaaagaacaa gccgaaatcg ttcgccgcgt cgagcaactc ttcgcctacg

2761 ccgacaccat agaaaaacag gtcaacaacg ccttagcccg cgtcaacaac ctgacgcaat

2821 ccatcctggc aaaagcgttc cgtggtgaac ttaccgccca gtggcgggcc gaaaacccgg

2881 atttgatcag cggagaaaac agcgccgccg cgttgctgga aaaaatcaaa gctgaacgcg

2941 cagctagcgg gggtaaaaaa gcctcacgta aaaaatcctg a

//

LOCUS mutant_M.EcoKI 2981 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 6)

DEFINITION .

FEATURES Location/Qualifiers

misc_feature 1..1590

/label="M"

CDS 1..1590

/label="Translation 1-1590"

misc_feature 1587..2981

/label="S"

CDS 1587..2981

/label="Translation 1587-2981"

ORIGIN

1 atgaacaata acTAActggt cgcgaagctg tggaagctgt gcgacaacct gcgcgatggc

61 ggcgtttcct atcaaaacta cgtcaatgaa ctcgcctcgc tgctgttttt gaaaatgtgt

121 aaagagacgg gtcaggaagc ggaatacctg cggaaggtt accgctggga tgacctgaaa

181 tcccgcatcg gccaggagca gttgcagttc taccgaaaaa tgctcgtgca tttaggcgaa

241 gatgacaaaa agctggtaca ggcagttttt cataatgtta gtaccaccat caccgagccg

301 aaacaaataa ccgcactggt cagcaatatg gattcgctgg actggtacaa cggcgcgcac

361 ggtaagtcgc gcgatgactt cggcgatatg tacgaagggc tgttgcagaa gaacgcgaat

421 gaaaccaagt ctggtgcagg ccagtacttc accccgcgtc cgctgattaa aaccattatt

481 catctgctga aaccgcagcc gcgtgaagtg gtgcaggacc cggcggcagg tacggcgggc

541 tttttgattg aagccgaccg ctatgttaag tcgcaaacca atgatctgga cgaccttgat

601 ggcgacacgc aggatttcca gatccaccgc gcgtttatcg gcctcgaact ggtgcccggc

661 acccgtcgtc tggcactgat gaactgcctg ctgcacgata ttgaaggcaa cctcgaccac

721 ggcggcgcaa tccgtctggg caacacTctg ggTagcgacg gtgaaaacct gccgaaggcg

781 catattgtcg ccactaaccc gccgtttggc agcgccgcag gcaccaacat tacccgcacc

841 tttgttcacc cgaccagcaa caaacagTtg tgctttatgc agcatattat cgaaacgctg

901 catcccggcg gtcgtgcggc ggtggtggtg ccGgataacg tgCtgtttga aggcggcaaa

961 ggcaccgaca tTcgtcgtga cctgatggat aagtgtcatc tgcacaccat tctgcgtctg

1021 ccgaccggta ttttttacgc Tcagggcgtg aagaccaacg tgctgttctt taccaaaggG

1081 acGgtggcga acccgAatca ggataagaac tgTaccgaTg aTgtgtgggt gtatgacctg

1141 cgtaccaata tgccgagttt cggcaagcgc acaccgttta ccgacgagca tttgcagccg

1201 tttgagcgcg tgtatggcga agacccgcac ggtttaagcc cgcgcactga aggtgaatgg

1261 agttttaacg ccgaagagac ggaagttgcc gacagcgaag agaacaaaaa caccgaccag

1321 catcttgcta ccagccgctg gcgcaagttc agccgtgagt ggatccgcac cgcaaaatcc

1381 gattcgctgg atatctcctg gctgaaagat aaagacagta ttgatgccga cagcctgccg

1441 gagccggatg tattagcggc agaagcgatg ggcgaactgg tacaggcgct gtctgaactg

1501 gatgcgctga tgcgtgaact gggggcgagc gatgaggccg atttgcagcg tcagttgctg

1561 gaagaagcgt ttggtggggt gaaggaatga gtgcgTAAaa attgccggag gggtgggtta

1621 tcgccccagt atctacggtc acaactctaa tccgaggagt aacgtataaa aaagagcagg

1681 caataaatta tctaaaagat gattatttgc ctcttatccg tgcgaacaat attcagaatg

1741 gcaagtttga tactacggac ttggtttttg ttcctaaaaa tcttgttaaa gaaaGtcaaa

1801 aaatatctcc tgaagatatt gttattgcaa tgtcatcagg gagcaaatcc gtagttggta

1861 aatccgcaca tcagcatcta ccatttgaat gtagtttcgg cgcattttgc ggtgtattac

1921 gtcctgaaaa acttatattt tctggtttta ttgctcattt cacaaaatct tctctttatc

1981 gaaacaaaat ttcatcactt tctgctggtg caaatattaa taatattaag ccggcaagct

2041 ttgatttgat aaatatacca atcccaccac ttgccgaaca aaaaatcatc gctgaaaaac

2101 tcgatacgct gctggcgcag gtagacagca ccaaagcacg ttttgagcaa atcccacaaa

2161 tcctgaaacg ttttcgtcaa gcggtattgg ggggcgcagt taatggaaaa ttgacagaaa

2221 aatggcgtaa ttttgagccg caacattctg tatttaagaa gttaaatttt gaatctatct

2281 taactgaatt acgtaatggt ctttcatcaa agccaaatga aagtggtgtt ggtcatccaa

2341 tactacgcat tagttctgta cgtgctggcc atgtagatca aaacgatatt cggtttctag

2401 aatgttcaga aagtgaacta aaccgccaca aattacaaga tggagatctt ttatttactc

2461 gctataacgg aagtttagaa tttgttggtg tttgtgggtt attgaaaaaa ttacaacatc

2521 aaaatttgct atatcctgat aaacttattc gagctcgatt aaccaaagat gctttaaccag

2581 aatatatcga aatatttttt tcatccccct cagcacgaaa tgcaatgatg aactgcgtga

2641 aaacaacttc tggtcaaaaa ggtatttcag gaaaagatat caaatcccaa gttgttttat

2701 tacctccagt aaaagaacaa gccgaaatcg ttcgccgcgt cgagcaactc ttcgcctacg

2761 ccgacaccat agaaaaacag gtcaacaacg ccttagcccg cgtcaacaac ctgacgcaat

2821 ccatcctggc aaaagcgttc cgtggtgaac ttaccgccca gtggcgggcc gaaaacccgg

2881 atttgatcag cggagaaaac agcgccgccg cgttgctgga aaaaatcaaa gctgaacgcg

2941 cagctagcgg gggtaaaaaa gcctcacgta aaaaatcctg a

//

LOCUS M.Eco0015 2954 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 7)

DEFINITION .

FEATURES Location/Qualifiers

misc_feature 1..1590

/label="M"

CDS 1..1590

/label="Translation 1-1590"

misc_feature 1587..2954

/label="S"

CDS 1587..2954

/label="Translation 1587-2954"

ORIGIN

1 atgaacaata acgatctggt cgcgaagctg tggaagctgt gcgacaacct gcgcgatggc

61 ggcgtttcct atcaaaacta cgtcaatgaa ctcgcctcgc tgctgttttt gaaaatgtgt

121 aaagagacTg ggcaggaagc ggaatacctg cggaaggtt accgctggga tgacctgaaa

181 tcccgcatcg gccaggagca gttgcagttc taccgaaaaa tgctcgtgca tttaggcgaa

241 gatgacaaaa agctggtaca ggcagttttt cataatgtta gtaccaccat caccgagccg

301 aaacaaataa ccgcactggt cagcaatatg gattcactgg actggtacaa cggtgcgcac

361 ggtaagtcgc gcgatgactt cggcgatatg tacgaagggc tgctgcagaa gaacgccaat

421 gaaaccaagt ctggcgcggg ccagtacttc accccgcgtc cgctgattaa aaccattatt

481 catctgctga aaccgcagcc acgtgaagtg gtacaggatc cggcagccgg tacggcgggc

541 tttttgattg aagccgaccg ctacgttaag tcgcagacta acgatctgga cgaccttgat

601 ggcgacacgc aggatttcca gatccaccgc gcgtttatcg gcctcgaact ggtgcccggc

661 acccgtcgtc tggcgctgat gaactgcctg ctgcacgata ttgaaggcaa cctcgaccac

721 ggcggcgcaa tccgtctggg caacaccctg ggcagcgacg gcgaaaacct gccgaaggcg

781 catattgtcg ccactaaccc gccgtttggc agcgccgcag gcaccaacat tacccgcacc

841 tttgttcacc cgaccagcaa caaacagttg tgctttatgc agcatattat cgaaacgctg

901 caccccggcg gtcgtgcggc ggtggtggtg ccggataacg tgctgtttga aggcggcaaa

961 ggtacggaca tccgtcgtga cctgatggat aagtgtcatc tgcacactat tctgcgtctg

1021 ccgaccggta ttttttacgc gcagggcgtg aagaccaacg tgctgttctt taccaaaggg

1081 acggtggcga acccgaatca ggataagaac tgcaccgatg acgtgtgggt gtacgacctg

1141 cggaccaata tgccgagctt tggtaaacgc acgccgttta ccgacgagca tttgcaaccg

1201 tttgagcgcg tgtttggcga agacccgcac ggcttaagcc cgcgcactga aggtgaatgg

1261 agttttaacg ccgaagagac ggaaattgcc gacagcgaag agaacaaaaa caccgaccag

1321 catcttgcta ccagccgctg gcgcaagttc agccgcgagt ggatccgcac cgcgaaatcc

1381 gattcgctgg atatctcctg gctgaaagat aaagacagta ttgatgccga caacctgccg

1441 gagccggatg tattagcggc agaagcgatg ggtgaactgg tacaggcgct gtctgaactg

1501 gatgcgctga tgcgtgaact gggggcgagc gatgaggcgg atgcgcagcg ccagttgctg

1561 gaagaagcat ttggtggggt gaaggaatga gtgctgggaa attgccggag gggtgggaac

1621 agattgaaat aggcgacatt gctgatgtca tctctggtgg gacaccaaaa tcgggagtcg

1681 ctgaaaattt tgcaccttcg ggtgagggag tcgcctggct tacgcctgcc gatttgagcg

1741 gttataaaga gaaatatatc tcacatggtg ccagagattt aactacacta ggctattcct

1801 cttgttcagc aaaactaatg cccaaaggca ctatactttt tagtagccgg gcaccgattg

1861 gatatgttgc tattgctgca aatgagattg cgactaacca gggtttcaaa agttttgctt

1921 ttccttcaga catttttcct gattatgcat attactttct ccgcaacatc agacatattg

1981 cagaggaaat gggcacagga acaactttta aagaaatttc tggtagttct gcgaaaacat

2041 taccatttgt tcttgtaccg tttgccgaac aaaaaatcat cgctgaaaaa ctcgatacgc

2101 tgctggcaca ggtagacagc accaaagcac gtcttgagca aatcccacaa atcctgaaac

2161 gttttcgtca ggcggttctg ggtgcagcgg tgagaggaaa actaactgaa gactggagag

2221 ataattcaag cctgtctggg tggagagaag gcaaacttgg ggagtttata aaaaaaccaa

2281 gttacggaac gtcatctaag tctaataaag aaggactcat tcctgtatta agaatgggca

2341 atcttcaggg aggaaagttg gactggacgg atctagtcta tacgtcagat accatagaaa

2401 ttgaaaagta taaacttgag tacaatgatg tattattcaa tcgtacaaat agtcctgaac

2461 ttgtcgggaa aacagccata tataaatcag agcaaccagc tatttatgca ggttatttga

2521 ttagggttca atgtttaacct gatttaaacc ctgactatct taactaccat ttaaatagca

2581 ttttggggag acaatattgc tattcagtta aatctgatgg ggtcagtcaa tcaaatatta

2641 atgcacagaa attaattgca tacccaataa ctgttccccc ccttcccgaa caagctgaaa

2701 tcgttcgccg cgtcgagcaa ctcttcgcct acgccgacac aatagaaaaa caggtcaaca

2761 acgccttagc ccgcgtcaac aatctgacgc aatccatcct ggcaaaagcg ttccgtggtg

2821 aactcaccgc ccagtggcgg gccgaaaacc cggaattaat cagcggtgaa aacagcgccg

2881 ccgcgttgct ggagaaaatc aaagctgaac gcgcagccag cgggggtaaa aaagcctcgc

2941 gtaaaaagtc ctga

//

LOCUS mutant_M.Eco0015 2954 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 8)

DEFINITION .

FEATURES Location/Qualifiers

misc_feature 1..1590

/label="M"

CDS 1..1590

/label="Translation 1-1590"

misc_feature 1587..2954

/label="S"

CDS 1587..2954

/label="Translation 1587-2954"

ORIGIN

1 atgaacaata acTAActggt cgcgaagctg tggaagctgt gcgacaacct gcgcgatggc

61 ggcgtttcct atcaaaacta cgtcaatgaa ctcgcctcgc tgctgttttt gaaaatgtgt

121 aaagagacTg ggcaggaagc ggaatacctg cggaaggtt accgctggga tgacctgaaa

181 tcccgcatcg gccaggagca gttgcagttc taccgaaaaa tgctcgtgca tttaggcgaa

241 gatgacaaaa agctggtaca ggcagttttt cataatgtta gtaccaccat caccgagccg

301 aaacaaataa ccgcactggt cagcaatatg gattcactgg actggtacaa cggtgcgcac

361 ggtaagtcgc gcgatgactt cggcgatatg tacgaagggc tgctgcagaa gaacgccaat

421 gaaaccaagt ctggcgcggg ccagtacttc accccgcgtc cgctgattaa aaccattatt

481 catctgctga aaccgcagcc acgtgaagtg gtacaggatc cggcagccgg tacggcgggc

541 tttttgattg aagccgaccg ctacgttaag tcgcagacta acgatctgga cgaccttgat

601 ggcgacacgc aggatttcca gatccaccgc gcgtttatcg gcctcgaact ggtgcccggc

661 acccgtcgtc tggcgctgat gaactgcctg ctgcacgata ttgaaggcaa cctcgaccac

721 ggcggcgcaa tccgtctggg caacaccctg ggcagcgacg gcgaaaacct gccgaaggcg

781 catattgtcg ccactaaccc gccgtttggc agcgccgcag gcaccaacat tacccgcacc

841 tttgttcacc cgaccagcaa caaacagttg tgctttatgc agcatattat cgaaacgctg

901 caccccggcg gtcgtgcggc ggtggtggtg ccggataacg tgctgtttga aggcggcaaa

961 ggtacggaca tccgtcgtga cctgatggat aagtgtcatc tgcacactat tctgcgtctg

1021 ccgaccggta ttttttacgc gcagggcgtg aagaccaacg tgctgttctt taccaaaggg

1081 acggtggcga acccgaatca ggataagaac tgcaccgatg acgtgtgggt gtacgacctg

1141 cggaccaata tgccgagctt tggtaaacgc acgccgttta ccgacgagca tttgcaaccg

1201 tttgagcgcg tgtttggcga agacccgcac ggcttaagcc cgcgcactga aggtgaatgg

1261 agttttaacg ccgaagagac ggaaattgcc gacagcgaag agaacaaaaa caccgaccag

1321 catcttgcta ccagccgctg gcgcaagttc agccgcgagt ggatccgcac cgcgaaatcc

1381 gattcgctgg atatctcctg gctgaaagat aaagacagta ttgatgccga caacctgccg

1441 gagccggatg tattagcggc agaagcgatg ggtgaactgg tacaggcgct gtctgaactg

1501 gatgcgctga tgcgtgaact gggggcgagc gatgaggcgg atgcgcagcg ccagttgctg

1561 gaagaagcat ttggtggggt gaaggaatga gtgctgggTA Attgccggag gggtgggaac

1621 agattgaaat aggcgacatt gctgatgtca tctctggtgg gacaccaaaa tcgggagtcg

1681 ctgaaaattt tgcaccttcg ggtgagggag tcgcctggct tacgcctgcc gatttgagcg

1741 gttataaaga gaaatatatc tcacatggtg ccagagattt aactacacta ggctattcct

1801 cttgttcagc aaaactaatg cccaaaggca ctatactttt tagtagccgg gcaccgattg

1861 gatatgttgc tattgctgca aatgagattg cgactaacca gggtttcaaa agttttgctt

1921 ttccttcaga catttttcct gattatgcat attactttct ccgcaacatc agacatattg

1981 cagaggaaat gggcacagga acaactttta aagaaatttc tggtagttct gcgaaaacat

2041 taccatttgt tcttgtaccg tttgccgaac aaaaaatcat cgctgaaaaa ctcgatacgc

2101 tgctggcaca ggtagacagc accaaagcac gtcttgagca aatcccacaa atcctgaaac

2161 gttttcgtca ggcggttctg ggtgcagcgg tgagaggaaa actaactgaa gactggagag

2221 ataattcaag cctgtctggg tggagagaag gcaaacttgg ggagtttata aaaaaaccaa

2281 gttacggaac gtcatctaag tctaataaag aaggactcat tcctgtatta agaatgggca

2341 atcttcaggg aggaaagttg gactggacgg atctagtcta tacgtcagat accatagaaa

2401 ttgaaaagta taaacttgag tacaatgatg tattattcaa tcgtacaaat agtcctgaac

2461 ttgtcgggaa aacagccata tataaatcag agcaaccagc tatttatgca ggttatttga

2521 ttagggttca atgtttaacct gatttaaacc ctgactatct taactaccat ttaaatagca

2581 ttttggggag acaatattgc tattcagtta aatctgatgg ggtcagtcaa tcaaatatta

2641 atgcacagaa attaattgca tacccaataa ctgttccccc ccttcccgaa caagctgaaa

2701 tcgttcgccg cgtcgagcaa ctcttcgcct acgccgacac aatagaaaaa caggtcaaca

2761 acgccttagc ccgcgtcaac aatctgacgc aatccatcct ggcaaaagcg ttccgtggtg

2821 aactcaccgc ccagtggcgg gccgaaaacc cggaattaat cagcggtgaa aacagcgccg

2881 ccgcgttgct ggagaaaatc aaagctgaac gcgcagccag cgggggtaaa aaagcctcgc

2941 gtaaaaagtc ctga

//

LOCUS pM0-Eco0015_R+M+S 6708 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 9)

DEFINITION .

FEATURES Location/Qualifiers

misc_feature 1..36

/label="pM0"

misc_feature 41..3553

/label="R"

CDS 41..3553

/label="Translation 41-3553"

misc_feature 3755..5344

/label="M"

CDS 3755..5344

/label="Translation 3755-5344"

misc_feature 5341..6708

/label="S"

CDS 5341..6708

/label="Translation 5341-6708"

ORIGIN

1 ggattaacaa tataagctga ccttcaagta ttgaatTGAC atgatgaata aatccaactt

61 cgaattcctg aagggcgtca acgacttcac ttatgccatc gcctgtgcgg cggaaaataa

121 ctacccggat gatcccaaca cgacgctgat taaaatgcgt atgtttggcg aagctacagc

181 gaaacatctt ggtctgttac tcaacatccc cccttgtgag aatcaacacg atctcctgcg

241 cgaactcggc aaaatcgcct tcgttgatga caacattctc tctgtattcc acaaattacg

301 ccgcattggt aaccaggcgg tgcacgaata ccataacgat ctcgacgatg cccagatgtg

361 cctgcgactc gggttccgcc tggctgtctg gtactaccgt ctggtcacca aagattatga

421 cttcccggtg ccggtatttg tgttgccgga acgtggcgaa aacctctatc accaggaagt

481 gctgacgcta aaacaacagc ttgaacagca ggcgcgagaa aaagcgcaga ctcaggcaga

541 agtcgaagcg caacagcaga agctggttgc cctgaacggc tatatcgcca ttctggaagg

601 caaacagcag gaaacagaag cgcaaaccca ggctcgcctt gcggcgctgg aagcacagct

661 cgccgagaag aacgcggagc tggccaaaca gaccgaacag gaacgtaagg cttaccacaa

721 agaaattacc gatcaggcca tcaagcgcac actcaacctt agcgaagaag agagtcgctt

781 cctgattgac gcgcaactgc gtaaagcagg ctggcaggcc gatagcaaaa ccctgcgctt

841 ctccaaaggc gcacgcccgg aacccggcgt caataaagcc attgccgaat ggccgaccgg

901 gaaagatgaa acgggtaatc agggctttgc ggattatgtg ctgtttgtcg gcctcaagcc

961 catcgcggtg gtagaggcga aacgtaaaaa tatcgacgtt cccggcaagc tcaatgagtc

1021 gtatcgctac agcaaatgct tcgataatgg cttcctgcgg gaaaccttgc ttgagcatta

1081 ctcaccggat gaagtgcatg aagctgtgcc ggagtatgaa actagctggc aggacaccag

1141 cggccaacaa cggtttaaaa tccccttctg ctactccacc aacgggcgcg aatatcgcgc

1201 agcaatgaag actaaaagtg gcatctggta tcgcgacgtg cgtgataccc gcaatatgtc

1261 gaaagcctta cccgagtggc atcgcccgga agagctgctg gaaatgctcg gcagcgaacc

1321 gcaaaaacag aatcagtggt ttgccgataa cccgggcatg agcgagctgg gcctgcgtta

1381 ttaccaggaa gatgccgtcc gcgcggtgga aaaggcaatc gtcaagggtc aacaagagat

1441 cctgctggcg atggcgaccg gtaccggtaa aacccgcacc gcaattgcca tgatgttccg

1501 cctgatccag tcccagcgtt ttaaacgcat tctcttcctt gtcgatcgcc gttctcttgg

1561 cgagcaggca ctgggggcgt ttgaagatac gcgtattaac ggcgacacct tcaacagcat

1621 tttcgacatt aaagggctga cggataaatt ccccgaagac agcaccaaaa ttcacgttgc

1681 caccgtacag tcgctggtga aacgcaccct acaatcagat gaaccgatgc cggtggcccg

1741 ttacgactgt atcgtcgtcg atgaagcgca tcgcggctat attctcgata aagagcagac

1801 cgaaggcgag ctgcagttcc gcagccagct ggattacgtt tctgcctacc gtcgcattct

1861 cgatcacttc gatgcggtaa aaatcgccct caccgccacc ccggcgctgc atacggtgca

1921 gattttcggc gagccggttt accgttatac ctaccgtacc gcggttatcg acggttttct

1981 gatcgaccag gatccgccga ttcagatcat cacccgcaac gcccaggagg gggtttatct

2041 ctccaagggc gaacaggtag agcgcatcag cccgcaggga gaagtgatca acgataccct

2101 ggaagacgat caggattttg aggtcgccga ctttaaccgt ggcctggtga tcccggcgtt

2161 taaccgcgcc gtctgtaacg agctcaccaa ctaccttgac ccgaccgggt cgcaaaaaac

2221 gctggtcttc tgcgttacca atgcccatgc cgatatggtg gtggaagagt tgcgtaccgc

2281 gttcaagaaa aagtatccgc aactggagca cgacgcgatc atcaagatca ccggtgatgc

2341 cgataaagac gcgcgcaaag tgcagaccat gatcacccgc ttcaataaag agcggctgcc

2401 caatatcgtg gtaaccgtcg acctgctgac aaccggcgtc gatattccgt cgatctgtaa

2461 tatcgtgttc ctgcgtaaag tacgcagccg tattctgtac gagcagatga aaggccgcgc

2521 cacgcgctta tgcccggacg tgaataaaac cagctttaag attttcgact gtgtcgatat

2581 ctacagcacg ctggagagcg tcgacaccat gcgtccggtg gtggtgcgtc cgaaggtgga

2641 actgcaaacg ctggtcaatg aaattaccga ttcagaaacc tataaaatca ccgaagcgga

2701 tggccgtagc tttgccgagc acagccatga acaattggtg gcgaagctcc agcgcatcat

2761 cggcctggcc acgtttaacc gtgaccgcag cgaaacgata gataaacagg tgcgtcgtct

2821 ggatgagcta tgccaggacg cggcgggcgt gaactttaac ggcttcgcct cgcgcctgcg

2881 ggaaaaaggg ccgcactgga gcgccgaagt ctttaacaaa ctgcctggct ttatcgcccg

2941 tctggaaaag ctgaaaacgg acatcaacaa cctgaatgat gcgccgatct tcctcgatat

3001 cgacgatgaa gtggtgagtg taaaatcgct gtacggtgat tacgacacgc cgcaggattt

3061 cctcgaagcc tttgactcgc tggtgcaacg ttccccgaac gcgcagccgg cattgcaggc

3121 ggtgatcaat cgcccgcgcg atctcacccg taaagggctg gtcgagctcc aggagtggtt

3181 tgaccgccag cattttgagg aatcttccct gcgcaaagca tggaaagaga cgcgcaatga

3241 agatatcgcc gcccggctga ttggtcatat tcgccgcgct gcggtgggcg atgcgctgaa

3301 accgtttgag gaacgtgtcg atcacgcgct gacgcgcatt aagggtgaaa acgactggag

3361 cagcgagcaa ttaagctggc tcgatcgttt agcgcaggcg ctgaaagaga aagtggtgct

3421 cgacgacgat gtcttcaaaa ccggcaactt ccaccgccgc ggcggtaagg cgatgctaca

3481 aagaaccttt gacgataatc tcgacaacct gcttgataaa ttcagcgatt atatctggga

3541 cgagctggcc tgacacgtat acacttcatc cttcaggctg cctctgcgtt ggctgcgctc

3601 gttcaccccg gtcacgtact tctgtacgct cccggggatt cactcacttg ccgccttgat

3661 gcaacctgaa tgattttgtg tatattaccc tcggcaattt cttcttctgc ggccccaaac

3721 aatgcgggcc gttgtttaat ttacggaact cacaatgaac aataacgatc tggtcgcgaa

3781 gctgtggaag ctgtgcgaca acctgcgcga tggcggcgtt tcctatcaaa actacgtcaa

3841 tgaactcgcc tcgctgctgt ttttgaaaat gtgtaaagag acTgggcagg aagcggaata

3901 cctgccggaa ggttaccgct gggatgacct gaaatcccgc atcggccagg agcagttgca

3961 gttctaccga aaaatgctcg tgcatttagg cgaagatgac aaaaagctgg tacaggcagt

4021 ttttcataat gttagtacca ccatcaccga gccgaaacaa ataaccgcac tggtcagcaa

4081 tatggattca ctggactggt acaacggtgc gcacggtaag tcgcgcgatg acttcggcga

4141 tatgtacgaa gggctgctgc agaagaacgc caatgaaacc aagtctggcg cgggccagta

4201 cttcaccccg cgtccgctga ttaaaaccat tattcatctg ctgaaaccgc agccacgtga

4261 agtggtacag gatccggcag ccggtacggc gggctttttg attgaagccg accgctacgt

4321 taagtcgcag actaacgatc tggacgacct tgatggcgac acgcaggatt tccagatcca

4381 ccgcgcgttt atcggcctcg aactggtgcc cggcacccgt cgtctggcgc tgatgaactg

4441 cctgctgcac gatattgaag gcaacctcga ccacggcggc gcaatccgtc tgggcaacac

4501 cctgggcagc gacggcgaaa acctgccgaa ggcgcatatt gtcgccacta acccgccgtt

4561 tggcagcgcc gcaggcacca acattacccg cacctttgtt cacccgacca gcaacaaaca

4621 gttgtgcttt atgcagcata ttatcgaaac gctgcacccc ggcggtcgtg cggcggtggt

4681 ggtgccggat aacgtgctgt ttgaaggcgg caaaggtacg gacatccgtc gtgacctgat

4741 ggataagtgt catctgcaca ctattctgcg tctgccgacc ggtatttttt acgcgcaggg

4801 cgtgaagacc aacgtgctgt tctttaccaa agggacggtg gcgaacccga atcaggataa

4861 gaactgcacc gatgacgtgt gggtgtacga cctgcggacc aatatgccga gctttggtaa

4921 acgcacgccg tttaccgacg agcatttgca accgtttgag cgcgtgtttg gcgaagaccc

4981 gcacggctta agcccgcgca ctgaaggtga atggagtttt aacgccgaag agacggaaat

5041 tgccgacagc gaagagaaca aaaacaccga ccagcatctt gctaccagcc gctggcgcaa

5101 gttcagccgc gagtggatcc gcaccgcgaa atccgattcg ctggatatct cctggctgaa

5161 agataaagac agtattgatg ccgacaacct gccggagccg gatgtattag cggcagaagc

5221 gatgggtgaa ctggtacagg cgctgtctga actggatgcg ctgatgcgtg aactgggggc

5281 gagcgatgag gcggatgcgc agcgccagtt gctggaagaa gcatttggtg gggtgaagga

5341 atgagtgctg ggaaattgcc ggaggggtgg gaacagattg aaataggcga cattgctgat

5401 gtcatctctg gtgggacacc aaaatcggga gtcgctgaaa attttgcacc ttcgggtgag

5461 ggagtcgcct ggcttacgcc tgccgatttg agcggttata aagagaaata tatctcacat

5521 ggtgccagag atttaactac actaggctat tcctcttgtt cagcaaaact aatgcccaaa

5581 ggcactatac tttttagtag ccgggcaccg attggatatg ttgctattgc tgcaaatgag

5641 attgcgacta accagggttt caaaagtttt gcttttcctt cagacatttt tcctgattat

5701 gcatattact ttctccgcaa catcagacat attgcagagg aaatgggcac aggaacaact

5761 tttaaagaaa tttctggtag ttctgcgaaa acattaccat ttgttcttgt accgtttgcc

5821 gaacaaaaaa tcatcgctga aaaactcgat acgctgctgg cacaggtaga cagcaccaaa

5881 gcacgtcttg agcaaatccc acaaatcctg aaacgttttc gtcaggcggt tctgggtgca

5941 gcggtgagag gaaaactaac tgaagactgg agagataatt caagcctgtc tgggtggaga

6001 gaaggcaaac ttggggagtt tataaaaaaa ccaagttacg gaacgtcatc taagtctaat

6061 aaagaaggac tcattcctgt attaagaatg ggcaatcttc agggaggaaa gttggactgg

6121 acggatctag tctatacgtc agataccata gaaattgaaa agtataaact tgagtacaat

6181 gatgtattat tcaatcgtac aaatagtcct gaacttgtcg ggaaaacagc catatataaa

6241 tcagagcaac cagctattta tgcaggttat ttgattaggg ttcaatgttt acctgattta

6301 aaccctgact atcttaacta ccatttaaat agcattttgg ggagacaata ttgctattca

6361 gttaaatctg atggggtcag tcaatcaaat attaatgcac agaaattaat tgcataccca

6421 ataactgttc ccccccttcc cgaacaagct gaaatcgttc gccgcgtcga gcaactcttc

6481 gcctacgccg acacaataga aaaacaggtc aacaacgcct tagcccgcgt caacaatctg

6541 acgcaatcca tcctggcaaa agcgttccgt ggtgaactca ccgcccagtg gcgggccgaa

6601 aacccggaat taatcagcgg tgaaaacagc gccgccgcgt tgctggagaa aatcaaagct

6661 gaacgcgcag ccagcggggg taaaaaagcc tcgcgtaaaa agtcctga

//

LOCUS M.EcoMII 2914 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 10)

DEFINITION .

FEATURES Location/Qualifiers

misc_feature 1..1620

/label="M"

CDS 1..1620

/label="Translation 1-1620"

misc_feature 1610..2914

/label="S"

CDS 1610..2914

/label="Translation 1610-2914"

ORIGIN

1 atggccaaag caccaactaa aaaagccaaa gcaaaaaaag gctttgaaga aacattatgg

61 gataccgcaa accaacttcg cggtagcgtt gagtcctccg aatacaagca cgtggtgtta

121 agcctcgtgt tcctgaaatt catcagcgat aagtttgaag cacgccgcaa aaaaatgatt

181 gccgatgggc aagctgactt ccttgagatg gaagtgttct accagcagga caacattttc

241 tacctgccgg aagaggcgcg ctggtcgttt atcaaacaaa atgccaaaca ggacgatatt

301 gcggttcgta ttgacaccgc cctctcgaca attgagaaac gtaacccaac cctgaaaggc

361 gcgctgccag acaactactt cagccgtcag aatctggaaa ccaaaaaact ggcgtcactg

421 attgatacca tcgacaacat cgaaacgctg gcacacgaaa ctgacgttga aacgttgtcg

481 aaagaagacc tggtcggacg cgtttacgaa tacttcctcg gtaagtttgc cgcaaccgaa

541 ggcaaaggcg gtggagagtt ctacacgccc aaatgtgtgg tcacgctgtt aactgaaatg

601 ctcgaaccct tccagggcaa aatttatgac ccgtgctgcg gctcggcagg gatgttcgtg

661 cagtcggtga agttcgtcga gagccatcag ggtaaaagcc gtgatatcgc gctgtatggt

721 caggaactga cagccacgac gtataaactg gcaaaaatga acctcgccat tcgcggtctt

781 tcagctaacc tcggcgaacg cccggcaaac actttcttta gcgaccagca cccggacctg

841 aaagccgact acattctggc gaacccgccg ttcaacctga aagactggcg caatgaagcc

901 gaattaaccg aagatccacg ctttgccggt tatcgtatgc cgccaaccgg taacgccaac

961 tacggctgga ttttgcatat gctctccaag ctgtcggcta acggcacagc aggttttgtg

1021 ctggcaaacg gttcgatgag ttctaacacc agcggtgaag gcgagatccg tgcacagatg

1081 atcgaaaatg atctgattga ctgcatgatc gccctacctg gacagttgtt ctacaccacg

1141 cagatcccgg tgtgtttatg gtttatgacc aaatcgaagg ctgccgaccc ggccaaaggt

1201 tatcgtgatc gtcagggcga gacgctgttt attgatgcgc gtaacctcgg caccatgatt

1261 agccgcacaa ctaaagagtt aacggcggaa gatattgcca caatcgccga tacctaccat

1321 gcctggcgca gcacgccgga agaactggct gcacggattg cgcgtggtga cagcaagctg

1381 gaaaaatatg aagaccaggc aggcttctgc aaagttgcga ccctgcaaga tattaaagat

1441 aacgactacg ttctgacacc gggccgctat gtgggtgcag ccgagcagga agaagacggc

1501 gtggcatttg agacgaaaat gcgtgaattg tcgaagacgt tgtttgagca gatgaagcag

1561 gcggaagaac tggatcgtgc gattcgccag aatctggagg cgctgggtta tggggagtaa

1621 gtggtgtata tatcgcttag gtgatatagc tgaattttca tacggaaaaa tgcctaaaaa

1681 ggagtttgtt ggtacaggtg actatgtcat tttttcagga tataaataca cagaaagcta

1741 ccctgaaaaa aatacttcgg cCggtgagtt aattgttgtt gcccgaggtg ttggtggaac

1801 tggcgatgtt aaaataacga ctagagattg ttatttaaca aatttgtcca ttaaaataaa

1861 tttggatgaa acaaaagtaa gaaaagaata tttttattat ctgtttctac agagtaattt

1921 acgatatctt gatagcggtt ctgctcaaag ccaaataacc atcaatgatc tagcgaatgt

1981 agaaatatct gctccttcgc taaatgttca agatttaata gtcaagagtc tcaaagcttt

2041 tgatgacaaa atcacaacac tttcatccat gaaccaaacc ttggaacaaa tgtcccaaac

2101 cctgttcaaa tcctggtttg tggattttga tccagtgatt gataacgctc tggatgcagg

2161 aaatccaatc ccggaagccc tgcaaactcg cgccaaattg cgtcaaaaag tacgtaatag

2221 tgcagatttt aaaccgcttc cggcggaaat tcgctcgctt ttcccaagtg aatttgaaga

2281 aacggagttg ggttgggtgc caaaggggtg gaaagaaggg acgcttccag agatagcttt

2341 tataaactcc acatcgtgga ctaataaaaa tcaaccagat tatatcaatt atgtagatct

2401 ctctaatgcg aaagatggca gaatatactc aattgaaaaa ttatcattta atgatgctcc

2461 aagtcgagct cgaagaatat taaaaaaaga tgatctgatt tttggccttg tcagacctgc

2521 taacagatca tttgcatatg tacatgttga tggtctaacg ggtagcacag gctttgccgt

2581 tattagagcc caaaagacga tatataaaaa tttcatttat tactttgtga cctatgataa

2641 aaatatagaa gaactagcga gaattgctga tggtggagca tatcctgcta taaaacctga

2701 tgatatttgt acattacctc taattattcc atcaataaat ataattgaga aatttgatga

2761 attaacaagc aatttcagag agatgatgaa taactcatta attcaaaacg aatatctcac

2821 caacctccga ggcaccttgc tcccgaaact catctccggc gagctatccc tggaagatct

2881 tccggatctc accaccgata cagaagccgc ataa

//

LOCUS M.EcoAI 3251 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 11)

DEFINITION .

FEATURES Location/Qualifiers

misc_feature 1..1482

/label="M"

CDS 1..1482

/label="Translation 1-1482"

misc_feature 1482..3251

/label="S"

CDS 1482..3251

/label="Translation 1482-3251"

ORIGIN

1 atggaataca acatgtctat cagctcagta atcaaatcat tacaagatat tatgcgcaaa

61 gatgccggtg tggacggaga tgcgcagcgt ctcggtcagc tctcctggct gctgtttttg

121 aaaatcttcg atgcccagga agaggcgctg gaactggagc aggataacta tcaatatccg

181 atcccacagc gttatttatg gcgcagttgg gccgcaaacg cgcagggcat taccggtgat

241 tccctgctgg aattcgttaa tgatgatctg ttcccggcgt tgaaaaacct cactgcgcct

301 atcgataaaa acccacgcgg ctacgtggtg aagcaggcgt tcagcgatgc ctataactat

361 atgaaaaacg gtacgctact gcgtcaggtg atcaacaagc tgaacgaaat tgactttacc

421 agcgccagcg aacgccatct gtttggtgat atttacgaac agatccttaa agatctgcaa

481 tctgcgggca atgctggcga attctatact ccacgcgccg tcactcgctt tatggtggat

541 cgcgttgatc cgaaactcgg cgaatccatt atggacccgg cctgcggtac gggcggtttt

601 cttgcctgcg catttgatca tgtaaagaac aaatacgtga agagcgtcgc cgatcatcag

661 acgctgcaac aacagatcca cggtgttgag aaaaaacagc ttccgcacct gctggcgacc

721 accaatatgc tgctgcacgg cattgaagta ccagtacaaa ttcgtcacga caacaccctg

781 aacaaaccgc tttcctcctg ggatgagcaa ctggatgtca ttgttaccaa cccgccgttt

841 ggtggcacgg aagaagacgg tattgagaag aactttccgg cagagatgca aacccgcgaa

901 acggcggatc tgttcctgca actgattgtg gaagtactgg cgaaaaacgg tcgtgcggcg

961 gtggtattgc cggatggcac actatttggc gaaggcgtta aaaccaaaat caaaaagcta

1021 cttaccgaag agtgcaacct gcataccatc gtgcgtttac cgaatggtgt gtttaacccc

1081 tataccggca ttaaaaccaa cctgttgttc tttaccaaag gtcagccaac caaagagatt

1141 tggttctatg agcatccgta tccggcgggc gtgaaaaact acagcaaaac caagccgatg

1201 aagtttgaag agtttcaggc ggagatcgac tggtggggta acgaggccga tggttttgcc

1261 agccgcgtcg agaatgagca agcgtggaaa gtcagcattg atgacgtgat tgcgcgtaac

1321 ttcaatctgg atattaaaaa cccacatcag gcggaaaccg tcagccatga tccggacgaa

1381 ctgttagcgc agtatgcaaa gcaacaggcg gagatccaga cgctgcgtaa tcaactgcgc

1441 gatattctcg gcgctgcgct gtctgtcaag gaggttaact gatgagtgtg gaaaagctga

1501 tcgttgatca tatggaaacc tggacctcgg cgttgcaaac ccgttccacc gccgggcgcg

1561 gcagttccgg taaaattgat ttgtatggca ttaagaaatt acgtgagctg attctggaac

1621 tggcagtgcg cggtaaactg gtgccgcagg atccgaacga tgaaccggcg tcggagctgc

1681 tgaagcgtat tgcggcggaa aaagcagagc tggtgaaaca ggggaaaatt aaaaagcaaa

1741 aaccactgcc ggaaattagc gaggaagaga agccgtttga attgccggat ggatgggagt

1801 ggacaacgct aactagaatt gcggaaataa atcctaaaat tgatgtcagt gatgatgagc

1861 aagaaatatc atttattcca atgccactca tatcaactaa atttgatggc tcgcatgaat

1921 ttgaaataaa aaaatggaaa gatgttaaaa aaggttatac acactttgct aatggtgata

1981 tcgctattgc aaaaataaca ccttgctttg aaaatagtaa agcagctatt ttttctggct

2041 taaaaaacgg cattggtgtt ggaacaacag aattacatgt tgcacgccct tttagcgata

2101 taattaatcg aaaatatctt cttttaaact ttaaatcacc taattttctt aagtctggtg

2161 aatcacaaat gactggttct gctggtcaga aacgtgttcc aaggtttttt tttgaaaata

2221 atcctattcc cttccctcca ttgcaagaac aagagcgcat tattattagg tttactcaat

2281 taatgtctct ctgcgaccaa ctggaacagc aatccctaac cagtctggac gcacatcagc

2341 aactggttga aaccctgttg ggaacactta cagacagcca aaacgtcgag gaactggctg

2401 aaaactgggc gcgtattagc gagcatttcg acacactatt taccactgaa gccagcgtgg

2461 atgcgttaaa acagaccatt ctgcaactgg ccgtaatggg taaacttgtg ccgcaggatc

2521 cgaatgacga acccgcctct gaactgctca aacgtattgc gcagaaaaaa gcgcaattgg

2581 tgaaagaagg gaaaataaaa aaacaaaaac cgttgccgcc aattagtgat gaggaaaaac

2641 cgtttgaact gccggaaggg tgggagtggt gtcgattggg ttctatttat aattttttaa

2701 atggatatgc ctttaaaagt gagtggttta cctctgtcgg tttacgttta ttgcggaatg

2761 ctaacattgc tcatggagtt accaattgga aagatgttgt acatatacca aatgacatga

2821 tatccgattt tgaaaattat attttgtcag aaaatgatat tgttatttca ttagacagac

2881 caattattaa taccgggtta aaatatgcta tcattagcaa atcagattta ccctgtttac

2941 tactccaacg agtagcaaaa tttaaaaatt atgcaaacac tgtgtctaac tccttcttga

3001 caatatggtt gcaatcttat ttcttcataa attcaattga tcctggaaga agtaatggag

3061 ttccacatat atctacgaaa cagttagaga tgacactatt tcctcttcta ccgcaaagtg

3121 aacaagatcg cattatttca aaaacggatg aattaataca aacctgtaat aaactgaaat

3181 atattatcaa aaccgccaaa caaacccaac tgcaccttgc agacgcactc actgacgcgg

3241 cgataaacta a

//

LOCUS M.EcoAO83I 3176 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 12)

DEFINITION .

FEATURES Location/Qualifiers

misc_feature 1..1470

/label="M"

CDS 1..1470

/label="Translation 1-1470"

misc_feature 1470..3176

/label="S"

CDS 1470..3176

/label="Translation 1470-3176"

ORIGIN

1 atgtctatca gttcagtaat caaatcatta caagatatta tgcgcaaaga tgccggtgtg

61 gacggcgatg cgcagcgtct cggtcagctc tcctggctgc tgtttttgaa aatcttcgat

121 gcccaggaag aggcgctgga actggagcag gataactatc aatatccgat cccacagcgt

181 tatttatggc gcagttgggc cgcaaacgct cagggcatta ccggtgattc cctgctggaa

241 ttcgttaatg atgatctgtt cccggcgttg aaaaacctca ctgcgcctat cgataaaaac

301 ccacgcggct acgtggtaaa gcaggcgttc agcgatgcct ataactacat gaaaaacggt

361 acgctactgc gccaggtgat caacaagctg aacgaaattg acttaccag cgccagcgaa

421 cgccatctgt ttggtgatat ttacgaacag atccttaaag atctgcaatc tgcgggcaat

481 gcgggcgaat tctatactcc acgcgccgtc actcgcttta tggtggatcg cattgatccg

541 aaactcggcg aatccattat ggacccggcc tgcggtacgg gcggttttct tgcctgcgcg

601 ttcgatcatg taaagaacaa atacgtgaag agcgtcgccg atcatcagac gctgcaacaa

661 cagatccacg gtgtcgagaa aaaacagctt ccgcacctgc tggcgaccac caatatgctg

721 ctgcacggca ttgaagtgcc tgtacaaatt cgtcacgaca acaccctgaa caaaccgctt

781 tcctcctggg atgagcaact ggatgtcatt gttaccaacc cgccgtttgg tggcacggaa

841 gaagacggta ttgagaagaa ctttccggca gagatgcaaa cccgcgaaac ggcggatctg

901 tttctgcaac tgattgtgga agtactggcg aaaaacggtc gcgcggcggt ggtattgccg

961 gatggcacgc tatttggcga aggcgttaaa accaaaatca aaaagctgct taccgaagag

1021 tgcaatctgc ataccatcgt gcgtttaccg aatggtgtgt ttaaccccta taccggcatt

1081 aaaaccaacc tgttgttttt taccaaaggt cagccaacca aagagatttg gttctatgag

1141 catccgtatc cggcgggcgt gaaaaattac agtaaaacca agccgatgaa gttcgaagag

1201 tttcaggcgg agattgactg gtggggtaac gaggccgatg gttttgccag ccgcatcgag

1261 aatgagcaag cgtggaaagt cagcattgat gacgtgattg cgcgtaactt caatctggat

1321 attaaaaacc cacatcaggc ggaaaccgtc agccatgacc cggacgaact gttagcgcag

1381 tatgcaaaac agcaggcgga gatccagacg ctgcgtaatc aactgcgcga tattcttggc

1441 gctgcgctgt ctggcaagga ggttaactaa tgaatgtgga aaagctgatc gttgatcata

1501 tggaaacctg gacctcggcg ttgcaaaccc gttccaccgc cgggcgcggc agttccggaa

1561 aaattgattt atatggcatt aagaaattac gtgagctgat tctggaactg gctgtgcgcg

1621 gtaaactggt gccgcaggat ccgaatgatg aaccggcgtc ggagctgctg aagcgtattg

1681 cggcggaaaa agccgagctg gtgaaacagg ggaaaattaa aaagcaaaaa ccactgccgg

1741 aaattagcga ggaagagaag ctatttgaat tgccggaggg atgggagtgg gtgcgatttg

1801 gtaatatcta tgaaatggag tacggtaata atttaccaca agaaaaaaga tctaattctg

1861 gtgaatataa tgtttatggc tccaatggag ttgtaggtac acacaatgaa gcatgtatta

1921 aatcaccatg tattattatt ggcagaaaag gttccgcagg agcattaaat ctttcaaatc

1981 agccagcctg ttgggtaaca gacgtagctt atagcactat acccccaatt gctatggttc

2041 ttgaatttgt ctttatacaa tttcatacat tgggcctcga taaattaggg aagggaatta

2101 agccaggctt gaatcgtaac gatgcttata gtttagtaat tgctattcca cctcgaagcg

2161 aacagaaagc catagtgtcg aaagtaaatg aattaatgtc cctatgcgac caactggaac

2221 agcaatcctt gaccagtctg gacgcacatc agcaactggt tgaaaccctg ttgggaacac

2281 ttgcagacag ccagaacgcc gaggaactgg ctgaaaactg ggcgcgtatt agcgagcatt

2341 tcgacacact ttttaccacc gaagccagcg tggatgcgtt aaaacagacc attctgcaac

2401 tggccgtaat gggtaaactt gtgccgcagg atccgaatga cgaacccgcc tctgaactgc

2461 tcaaacgaat tgcgcaggaa aaagctcaac tggtgaaaga agggaaaata aaaaaacaaa

2521 aaccgttgct gccaattagc gatgaggaaa aaccgtttga actgcctaat ggatgggaat

2581 ggtgcaggct cggagagctt attgattcca ttgatgctgg gtggagtcct gcatgttctt

2641 ctgaaccagc cgctccagga gaatgggggg tgttaaaaac gaccgcagtt caatctcttg

2701 aatatagaga gtacgaaaac aaggcgctcc ctaaaaataa agccccaagg ccacaactag

2761 aagtaaaagc aggagatatt cttataacaa gagctggtcc taaaaatcgg gttggtattt

2821 catgccttgt tgaaaacaca agagaaaatt taatgatttc agataaaata atcagatttc

2881 atttgatatc agaggatatc tccgagaaat acatttcctt atgtctaaac tatgggttta

2941 cttcaacata tcttgaaaac tccaaatctg gcatggcaga aagtcagatg aatatttcac

3001 aagatatttt aaaaatggca cctatagcta tcccgactac acatgaacaa ttaaaaataa

3061 cagataaaat aaatgaaatg atggattatt ttataactct aaagagtcaa attcaatctg

3121 cccaacaaac ccaactgcac cttgcagacg cactgactaa cgcggcgata aactaa

//

LOCUS M.EcoMIII 2875 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 13)

DEFINITION .

FEATURES Location/Qualifiers

misc_feature 1..1638

/label="M"

CDS 1..1638

/label="Translation 1-1638"

misc_feature 1628..2875

/label="S"

CDS 1628..2875

/label="Translation 1628-2875"

ORIGIN

1 atggtagaac tggaatttag agacaaaacc aaagccctga tcgacagcct gaaaagcatc

61 tgtgcgaact acggcctggg caacgacggt aacgaattca aaattatcac tcaggcattc

121 ctgtacaaat tcctgaacga caaattcgct tttgaggcca aacagaaaga taaaagtatc

181 gccagcgccg aaagctggga agatgcgctc agcgcaatga gcgaagatca actgaaaaag

241 ctgcaacagc ggatggcccc ggataccgcc cgcctgaagc cgcaccattt tattcgttac

301 ttgtataacc gacaaaacgc agcggacttt gccagaactt ttgacgacac cctgatggac

361 atcgccgcca ccaacaacga cgtcttcgcg gtcaaaaccg acggcggcgc gaaagtggtg

421 ctgtttgagc gcctcagcca gtacatcgcc gacgaatcaa agcgcgatga tttctgccgc

481 gccatcatca acaagctggc cgatttcagc ttcgaacgca tcttcacgca gaaattcgac

541 ttctacgcca ccatctttga atacctgatc aaagactaca acagcaactc cggcggcaaa

601 tacgcggaat actacacgcc ccacgccgtg gcgcgcatca tggcggaaat cctggtgccg

661 aaagcgcagc agggcgtggt gcgcaacgta agctgttacg acccgtccgc cggttccggc

721 acgctgctga tgaacgtggc gcacgccatt ggggaagatc gttgcagcat ctttgcccag

781 gatatctcgc agaaatcctc cagcctgctg cgtctgaacc tgatcctcaa caacctggtg

841 cattcgatcc ctaacgttat ccagggcaac actattctgc atccgttcca caaggacggc

901 ggcgcgctga aacgctttga ctacatcgtc tccaacccgc cgtttaagat ggatttcagc

961 gactttcgtg acgcgctgga cagcaaggaa aaccagcaac gtttctttgc cgggataccg

1021 aaaatcaaag ccaaagccag ggacaaaatg gagatttacc agttgttcct gcaacacatc

1081 attttctcgc tgaaaccggg tggcaaggcg gcggtcgtcg tgccgaccgg gtttatcacc

1141 gcgcaatcgg gcattgataa aggcattcgc gaacatctgg tgcaaaacaa gatgctggcg

1201 ggcgtggttt ccatgccgtc gaatatcttc gccaccaccg gtaccaacgt gtcgatcctg

1261 tttattgacg ccagcaacaa agagaaagtg gtgctgatcg acgcctcgaa tttaggtgaa

1321 aaggtcaaag acggcaaaaa ccagaaaacg gtgctgacgg agtgcgaaga aaaacggatc

1381 tgcgaagcgt ttaacaacaa gtggagcgaa gaggatttct cggtggtggt cagctatgac

1441 gacatcgccg cgaaaaatta ctcgttcagc gccggacagt attttgatgt gaaaatcgaa

1501 tacaccgaca tgacgccgga acagtttgcc gcgaagatga aaggatttac ggagaatctg

1561 aataatctct ttgaacagtc gcgcgagtta gaagtgcaaa taaaaatgca gatggcagat

1621 atcaaatatg aaatttaatt atctgaaaat taaagatgta tgtgattttg tcggaggaag

1681 tcaacctcca aaatctcaat ttatttatgt atctaaacct ggatatgtca ggttgatcca

1741 gacgcgtgat tacaaaacgg atgcatttcc cacgtatata ccaatatcgt cgacgaagaa

1801 attttgtgat gaattcgata ttatgattgg taggtatggt cccccaattt ttcaaatatg

1861 tcgcggattg aaaggagcat ataacgttgc cttgctgaag gttataccta aagaaggtgt

1921 aagtcgtgat tttttatact attttttgaa gcaagactct gttttccaat atgttgataa

1981 attatctgcc agaaccggag gacaaactgg agttgatcta gtttctttaa aagaatatcc

2041 agtcaggata cctgaagaaa ttgaatgtca agaaaaattg gtgactattc ttagtgttat

2101 tgataaaaaa atcgccctca acaaccgcat caacacggaa ctggaagcga tggcgaaaac

2161 actgtatgac tactggtttg tacagtttga tttccctgat gccaacggaa aaccgtataa

2221 aacctccggc gggaagatgg agtataacgc cacgctgaaa cgggaaattc cggcaggttg

2281 gaatgactcc attcttggta agtttattga gttagatcgt ggcgttactt atagcaaaga

2341 ggatgttcga actcaggatg ataaagatac gataggcatt cttcgcgcta caaatgtgac

2401 gggaaataat gttgatattg atgatttagt cttcattcct tctagtagag taaacgttaa

2461 tcaaatgcta aataaatttg acatattgat cgtgatgtca agtggtagta aggagcatgt

2521 agggaagaat ggcgtgtatt atttcgaaaa aaaacatgca tttggcgcat tttgttcaaa

2581 aattacaccc gtcagaaaat acagatattt tataaataca ttcctgcaat cgaaatggtt

2641 taagtcttat ataaacaatc agtgcttggg aactaatatt aataacctga ccaatacgca

2701 tatcactaat tgtgaaatca tatgccccac tcctgatgtt gtggcgttat tcgagaataa

2761 aatgatgcca atttataata aactggcatc aaacactcaa gagaatagtc atttaatcca

2821 actccgcgac tggctcctcc ctctacttat gaacggacag gtcacggtca aataa

//

LOCUS M.Eco1167 3794 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 14)

DEFINITION .

FEATURES Location/Qualifiers

misc_feature 1..2439

/label="M"

CDS 1..2439

/label="Translation 1-2439"

misc_feature 2451..3794

/label="S"

CDS 2451..3794

/label="Translation 2451-3794"

ORIGIN

1 atggctatca aaaaaaccga actctattct tccctgtggg caagctgtga cgaactgcgt

61 ggcgggatgg atgccagcca gtacaaagac tatgtactga ccctgctgtt tatgaagtac

121 gtttccgata agtataaagg cgatccttac gggatgatcg tgatacccaa aggagccagc

181 tttgacgata tggttgcgct gaaaaatgac aaagagatcg gcgacaaaat caacaaaatc

241 atccacaagc tggcggaaga gaacggcctg aagggcgtca tcgacgaagc cgactttaac

301 gacgaagata agctcggcaa aggcaaggaa atgattgacc gcctgagcaa gcttgtcggt

361 atctttgaag ggttaaatct ctccagcaac cgggccgaag gcgacgatct gctgggcgat

421 gcctatgaat atctgatgcg tcacttcgca accgaatccg gtaagagtaa aggacagttt

481 tacactccag cggaggtgtc acgtattctg gcgaaggtga ttggtattac tcccgatact

541 ccgcaagatg caacggttta tgacccaacc tgtggctctg gttcgctgct gttgaaagtg

601 aatgatgaag cccggcgcgg actgtcgatt tttggccagg agatggataa cgctaccagt

661 gcgctggcgc ggatgaacat gattctgcat aacaatgcca ccgcgaaaat ctggcagggc

721 aatacgctga gcgatccgca gtggaaagag gcgaacggca aactgaaagc cttcgatttt

781 gccgtggcga acccgccttt ttccaataaa aactggacca acgggctgac gccgaaaaaa

841 gatccgtttg aacgcttcgg ctggggtatt ccgccggaaa aaaacggcga ttatgctttt

901 ttgttgcata tcattaaaag cctgaaaagt accggcaaag gcgcggtgat cctgccccac

961 ggcgtactgt ttcgcggtaa cgccgaagcg aatatccgtg aaaacctaat caagcagggc

1021 tatatcaaag gcgttattgg tttgccagcc aacctgtttt acggcaccgg catcccggcg

1081 tgcatcattg tgattgataa agagcacgcc cacagccgca aaggcatttt tatgatcgac

1141 gccagccggg gctttatcaa agatggcaac aagaaccgcc tgcgtagccg agatattcac

1201 cgcattgtgg acgtgttcaa ccatcagcga acggtacctg gctacagccg gatggtgccc

1261 tccagcgaga tcgccggaaa tgattacaac ctgaatattc ctcgctatat cgagagcggc

1321 gagccggaag atttacacga tctgaccgcg cacttacagg gcggtattcc ggcacgcgat

1381 gtggatgctt tgcaagatta ctggcgggta ttccctgccc tgcgcaacgt gctgtttgct

1441 gacgaccgtc ccggttactg tcgggcacag gttaacgcgc aacaggtgaa accgatcatt

1501 ctggcgcatc aggagtttaa ggattttgcc acccgcagcc tgctaccgtt taaagcgtgg

1561 gtgaaagagg caagtctgga agagattcgc aaaggtgata aacccaaagc gctaatccac

1621 gatatcagcg aaatgctatt ggcgcagtac gccaacagtg agttgctgaa caaatacagc

1681 gtgtaccaga tcctgatgga ttactggact gatgtcatgc aagacgatgt gtatgcgatt

1741 atgcaggatg gctggcaagc cgccgcgcaa atccgcgagc ttcagccggt gaaaggcaaa

1801 gatggcaaaa acatctggaa agaaacgcac gatttcgagt ttaccaaacg gcgctacaag

1861 gccgatgtgc tgccccgttc gctagtggaa attcgctgtt tcccggaact gctggatgcg

1921 ctgaaccgcg cgcaggagcg cagtgaagaa gccagtcgtc agctagtgga attcatcgag

1981 gagcaggcag gggaagagga tctgctggct gaggcgaaaa acgataaaga taaggtgacg

2041 caaaagctgg tgaatgcccg tctggcacag ttgaagaaaa cccctgccga cccggatgag

2101 ctggcagtgc tgaaccgctg tctggcgctg attaaggccg aggccagtgc gaaaaaagcg

2161 ctgaaagccg cacaggaagc gttggatcga gcggtattta aacattatcc cacgctggat

2221 gaagcagcga ttaaaacgct ggtagtgcag gataagtggc tggcaacgtt gcaggcaggg

2281 atcaaagcgg agatagagcg tattacccag caactggcgt cgcgcgtaaa ggagctggaa

2341 gagcgttatg ctgaaccgtt gccagcactt gaagccgccg ttgaggcgtt gagcgagaaa

2401 gtggccgggc atttgcgggc gatggggctg gagtggtgat ggtggattta atggtgaatg

2461 agcaacggtc gggtacggtg gggaaaggtg tgcctgcggg gtataagctg actgagatgg

2521 agatgattcc tgaggattgg gttgtatcca cgatattaaa cctgacaacc aacataattg

2581 attatcgcgg cagaactccc aaaaaacttg gaatggattg gggggatggg gatatcgttg

2641 ccctttctgc tgctaatgtt aaaaaaggat atatagattt atctactgaa tgctactttg

2701 gttccgaaga actatataag cgctggatga catccggtca tccccaaaaa ggggacatag

2761 ctttcacaat ggaagcccct ctggggaatg cagcatctat tcctgataac agaaaatata

2821 ttcttagcca gagaacaatt cttttacaaa tagatagaga gaatttttct ccatcactaa

2881 tactccaaat cttattatca gagcgttttc aatcatatat ttctgaaagc gcgactggtt

2941 caacggccca agggattaaa cgttcagttt tagagaagtt atacatatct ataccgaaaa

3001 gtatcgtaga gcaaaaggct attgctaatg tattaaccaa tgttgatagc ttgattctat

3061 ccctagaaaa attactctca aaaaaacagt ccatcaaaac tgccacaatg cagcaactac

3121 tgactggtaa aacccgtttg ccacagtttg cgttgcgcaa agatggctct gctaagggat

3181 ataaaaagag cgagttgggg gagattcctg aagattggaa ttgtattaat cttggtgagc

3241 taggtaattg cattattggg ttaacttaca aacctgaaga tacatctgat tatggtacat

3301 tggtattacg ctcatccaac attcaaaata atcacttagc ttatgatgat aacgtcttcg

3361 tcagcataga accacctgag cgcacaattg tgaaagaagg tgatatcttg gtatgtgtaa

3421 gaaatggcag tcgccaactt attggcaaat gcgcattaat tgacaaaaat gcgcagggct

3481 cagcattcgg tgcatttatg tcagtattcc gaactgatat atatggattt acatttcacc

3541 aattccagtc tgatattatt caaaagcaaa tcgctgaagt tatgggtgcc actatcaatc

3601 agataactaa caaagatatg ttgggcttta aaatcccatt acctgaaaat acagaagaac

3661 aagccgccat cgccgccatc ctctccgata tggacaagga aatccaaatc ctgcaacagc

3721 gtctggacaa aacccgccag cttaagcaag ggatgatgca ggaactgtta accgggaaaa

3781 tccgcttaat atag

Appendix V: p1065 GenBank DNA sequence file

LOCUS p1065 9705 bp ds-DNA circular 01-NOV-2021 (SEQ ID NO: 15)

DEFINITION .

KEYWORDS "Plasmid_p1065:P2 test construct for methylation

protection""Resistance:Spc"

FEATURES Location/Qualifiers

misc_feature 1..92

/label="Promote"

CDS 93..881

/label="aadA"

terminator 1066..1153

/label="rrnBT2 term"

misc_feature 1164..1197

/label="J23100"

misc_feature 1205..1218

/label="rbsJ6110"

misc_feature 1225..1941

/label="sGFP"

misc_feature 1946..1997

/label="terminator BBa_K864600"

misc_feature 2936..2949

/label="Eco0015"

misc_feature 2936..4322

/label="Test Block"

misc_feature 2971..2984

/label="Eco1167"

misc_feature 3010..3020

/label="EcoMIII"

misc_feature 3053..3063

/label="EcoMII"

misc_feature 3103..3115

/label="EcoKI"

misc_feature 3149..3162

/label="EcoAI"

misc_feature 3179..3193

/label="EcoAO83I"

misc_feature 4038..4052

/label="EcoAO83I"

misc_feature 4083..4096

/label="EcoAI"

misc_feature 4141..4153

/label="EcoKI"

misc_feature 4186..4196

/label="EcoMII"

misc_feature 4226..4236

/label="EcoMIII"

misc_feature 4265..4278

/label="Eco1167"

misc_feature 4309..4322

/label="Eco0015"

terminator 5716..5787

/label="rrnB T1 terminator"

CDS complement(5814..6692)

/label="araC"

promoter 6719..7003

/label="araBAD promoter"

misc_feature 7004..8559

/label="P4"

cds 7039..7535

/label="delta (P2 activation)"

cds 7609..8181

/label="psu"

misc_feature 8246..8472

/label="Essential P4 packaging signal"

misc_feature 8920..9658

/label="CloDF13 ori"

ORIGIN

1 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat

61 aaatgcttca ataatattga aaaaggaaga gtatgaggga agcggtgatc gccgaagtat

121 cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg

181 ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt

241 tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc

301 ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca

361 ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg

421 gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg

481 atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg

541 cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct

601 taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt

661 tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg

721 actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg

781 cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg

841 tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataatgtcta acaattcgtt

901 caagccgagg ggccgcaaga tccggccacg atgacccggt cgtcggttca gggcagggta

961 ccaggcacgc ctaaccgtca gtgagattgg atgagtgaac gatattgatc gagaagagcc

1021 ctgcgcagcc gctgccgtgc ctgcaggaag caacggcccg gagggtggcg ggcaggacgc

1081 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt

1141 gcgtttctac aaactctgct agcttgacgg ctagctcagt cctaggtaca gtgctagctc

1201 tagagaaaga ggcgaattac tagtatgcgt aaaggcgaag aactgttcac gggcgtagtt

1261 ccgattctgg tcgagctgga cggcgatgtg aacggtcata agtttagcgt tcgcggtgaa

1321 ggtgagggcg acgcgaccaa cggcaaactg accctgaagt tcatctgcac caccggtaaa

1381 ctgccggtgc cttggccgac cttggtgacg acgttgacgt atggcgtgca gtgttttgcg

1441 cgttatccgg accacatgaa acaacacgat ttcttcaaat ctgcgatgcc ggagggttac

1501 gtccaggagc gtaccatttc cttcaaggat gatggctact acaaaactcg cgcagaggtt

1561 aagtttgaag gtgacacgct ggtcaatcgt atcgaattga agggtatcga ctttaaagag

1621 gatggtaaca ttctgggcca taaactggag tataacttca acagccataa tgtttacatt

1681 acggcagaca agcaaaagaa cggcatcaag gccaatttca agattcgcca caatgttgag

1741 gacggtagcg tccaactggc cgaccattac cagcagaaca ccccaattgg tgacggtccg

1801 gttttgctgc cggataatca ctatctgagc acccaaagcg tgctgagcaa agatccgaac

1861 gaaaaacgtg atcacatggt cctgctggaa tttgtgaccg ctgcgggcat cacccacggt

1921 atggacgagc tgtataagta atgagttgtt cagaacgctc ggtcttgcac accgggcgtt

1981 ttttctttgt gagtccaaat tacagctttc aaaagatact tacgatgatg atttagataa

2041 tttattggcg caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc

2101 agatgctatt ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct

2161 atcagcttca atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc

2221 tttagttcga caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa

2281 cggatatgca ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa

2341 accaatttta gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga

2401 tttgctgcgc aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg

2461 tgagctgcat gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg

2521 tgagaagatt gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg

2581 tggcaatagt cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa

2641 ttttgaagaa gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa

2701 ctttgataaa aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta

2761 ttttacggtt tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc

2821 agcatttctt tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg

2881 aaaagtaacc gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttccatc

2941 gattgcttct taccgcgtgc gagatagcca ttcatgaatc gttcagcctg cagtcaggtc

3001 tattagttta aaggtcagtt gtaacgttga tgcccctaag aacctctcgg tggtaccatt

3061 gtgcgacgca agcgattaca ctcctgtcac atcataatcg ttaacacgtc agtgcgtttg

3121 ctattcaggg cttgaccaac actggattga gataactagt catggtaaac actccacagg

3181 aattcttaca tgcttgatgc tacggcgatt cttggagagc cagcagcgac tgcaaatgtg

3241 agatcagagt aatattagca agcgataagt ccctaactgg ttgtggcctt ttgtagagtg

3301 aacttcataa catatgctgt ctcaggcacg tggatggttt ggacaaatca gattcaagtc

3361 tgatcaacct tcatacagat ctagagtcta aagcagtgat ctcaaaatac taggtaacta

3421 gagggactgc gacgttctaa acgttggtcc gtcagaagcg ccatccagga tcacgttacc

3481 ccgaaaaaaa gatatcagga gctctcctcc tcagaaacta caggactaac cttcctggca

3541 accgggaggt gggaatccgt cacatatgag aaggtatttg cccgataatc aatactgcct

3601 taagccggct tgccctttct gcctgtagat ccattggact ggtgccaacg cgcaggcata

3661 gttcgaggag aattatccgg gggcaatgac aaccagcatc tcgggtcttg cccaacccgt

3721 ctacacgctc cttaactctg gcaggcaatt aaagggaacg tatatataac gcaaagaagc

3781 tggaaaattg gcgagagaat cttctctctg tctatcgaag aatggccacg cggtggcaac

3841 cgtcatgcta gcgtgcgggg tacacttgct aaccatttgg gacacgggac actcgctgtt

3901 ttcgaaatta ccctttatgc gcgggtattg aaccacgctt atgcccagca tcgttacaag

3961 cagactcata ctagatgtat tatgcccgcc atgcagacga aaccagtcgg agattaccga

4021 gcattctatc acgtcgggga attcttacat gccgaccata agtgagctac tggagccgag

4081 gggagataac tagtcagctt ttcacttaaa gtattatgca cgacagggtg cgtgtacgtt

4141 aacacgtcag tgccatgtaa acctgttata acttacctca gttaggtacc attgtgttgg

4201 aagtgtggct agatcttagc tttttaaagg tcagttttcc ggtgccacgc gatggaacga

4261 gccattcatg aatcgttcta ttgttatagc gaatcagcgg gaacgcaacc atcgattgct

4321 tctttatgat gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc

4381 aaaatatttc ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa

4441 tggagagatt cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg

4501 ggataaaggg cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat

4561 tgtcaagaaa acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag

4621 aaattcggac aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt

4681 tgatagtcca acggtagctt attcagtcct agtggttgct aaggtggaaa aagggaaatc

4741 gaagaagtta aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt

4801 tgaaaaaaat ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt

4861 aatcattaaa ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct

4921 ggctagtgcc ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa

4981 ttttttatat ttagctagtc attatgaaaa gttgaagggt agtccagaag ataacgaaca

5041 aaaacaattg tttgtggagc agcataagca ttatttagat gagattattg agcaaatcag

5101 tgaattttct aagcgtgtta ttttagcaga tgccaattta gataaagttc ttagtgcata

5161 taacaaacat agagacaaac caatacgtga acaagcagaa aatattattc atttatttac

5221 gttgacgaat cttggagctc ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa

5281 acgatatacg tctacaaaag aagttttaga tgccactctt atccatcaat ccatcactgg

5341 tctttatgaa acacgcattg atttgagtca gctaggaggt gactgaccgg ctgataaatt

5401 tctttgaatt tctccttgat tatttgttat aaaagttata aaataatctt gttggaacca

5461 ttcaaaacag catagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca

5521 ccgagtcggt gctttttttg atacttctat tctactctga ctgcaaacca aaaaaacaag

5581 cgctttcaaa acgcttgttt tatcattttt agggaaatta atctcttaat ccttttatgc

5641 gatcgcgggc catcgccctg atagactata aacgcagaaa ggcccacccg aaggtgagcc

5701 agtgtgactc tagtagagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc

5761 tttcgactga gcctttcgtt ttatttgatg cctggagatc cttactcgag ttattatgac

5821 aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc gctcgggctg

5881 gccccggtgc atttttaaa tacccgcgag aaatagagtt gatcgtcaaa accaacattg

5941 cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc ctggctgata

6001 cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa aagatgtgac

6061 agacgcgacg gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa attgctgtct

6121 gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat cggtggatgg

6181 agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag atttatcgcc

6241 agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg

6301 ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc aaatattgac

6361 ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaaaccca ctggtgatac

6421 cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa

6481 atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc ctgaccgcga

6541 atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa aaaatcgaga

6601 taaccgttgg cctcaatcgg cgttaaaccc gccaccagat gggcattaaa cgagtatccc

6661 ggcagcaggg gatcattttg cgcttcagcc atacttttca tactcccgcc attcagagaa

6721 gaaaccaatt gtccatattg catcagacat tgccgtcact gcgtctttta ctggctcttc

6781 tcgctaacca aaccggtaac cccgcttatt aaaagcattc tgtaacaaag cgggaccaaa

6841 gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt ccacattgat

6901 tatttgcacg gcgtcacact ttgctatgcc atagcatttt tatccataag attagcggat

6961 cctacctgac gctttttatc gcaactctct actgtttctc cataattttc ttaacctgaa

7021 gtgacgagga gccggaaatt tactgtccgt cgtgtggaca tgttgctcac acccgtcgcg

7081 cacatttcat ggacgatggc accaagataa tgattgcaca gtgccggaat atttatgct

7141 ctgcgacatt tgaagcgagt gaaagctttt tctctgacag taaagattca ggaatggaat

7201 acatttcagg caaacagaga taccgcgatt cactgacgtc agcctcctgc ggtatgaaac

7261 gcccgaaaag aatgcttgtt accggatatt gttgtcggag atgtaaaggc cttgcactgt

7321 caagaacatc gcggcgtctg tctcaggaag tcaccgagcg tttttatgtg tgcacggatc

7381 cgggctgtgg tctggtgttt aaaacgcttc agaccatcaa ccgcttcatt gtccgcccgg

7441 tcacgccgga cgaactggca gaacgcctgc atgaaaaaca ggaactgccg ccagtacggt

7501 taaaaacaca atcatattcg ctgcgtctgg aatgagggct gccggttaac accggccgtc

7561 gccgcacacc gtatttttat tcttcagcat gatgagaaag agataacgat ggaaagcaca

7621 gccttacagc aggcctttga cacctgtcag aataacaaag cagcatggct gcaacgcaaa

7681 aatgagctgg cagcggccga acaggaatat ctgcggcttc tgtcaggaga aggcagaaac

7741 gtcagtcgcc tggacgaatt acgcaatatt atcgaagtca gaaaatggca ggtgaatcag

7801 gccgccggtc gttatattcg ttcgcatgaa gccgttcagc acatcagcat ccgcgaccgg

7861 ctgaatgatt ttatgcagca gcacggcaca gcactggcgg ccgcactggc accggagctg

7921 atgggctaca gtgagctgac ggccattgcc cgaaactgtg ccatacagcg tgccacagat

7981 gccctgcgtg aagcccttct gtcctggctt gcgaagggtg aaaaaattaa ttattccgca

8041 caggatagcg acattttaac gaccatcgga ttcaggcctg acgtggcttc ggtggatgac

8101 agccgtgaaa aattcacccc tgcgcagaac atgatttttt cgcgtaaaag tgcgcaactg

8161 gcatcacgtc agtcagtgta aaattccccg aaaatccgcc cgtttttact gaaaaaagcc

8221 atgcatcgat aaggtgcatg gctttgcatg cgttttcctg cctcattttc tgcaaaccgc

8281 gccattcccg gcgcggtctg agcgtgtcag tgcaactgca ttaaaaccgc cccgcaaagc

8341 gggcgggcga ggcggggaaa gcaccgcgcg caaaccgaca agttagttaa ttatttgtgt

8401 agtcaaagtg ccttcagtac atacctcgtt aatacattgg agcataatga agaaaatcta

8461 tggcctatgg tccaaaactg tcttttttga tggcactatc ctgaaaaata tgcaaaaaat

8521 agattgatgt aaggtggttc ttgtcagtgt cgcaagatcg ggccccattc tacattttta

8581 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcagcgc

8641 gggtgtggtt gtgcttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt

8701 atcaacttga aaaagtggca ccgagtcggt gctttttttg gcgctgccat cttggcggcc

8761 taaggcgatg ccccctcgac ctcgatcagg gaggcgttca ggacgactca caaagaaagc

8821 cgggcaatgc ccggcttttt ccacgcctcc tgggctgact tcaggtgcta catttgaaga

8881 gataaattgc actgaaatct agagcggttc agtagaaaag atcaaaggat cttcttgaga

8941 tccttttttt ctgcgcgtaa tcttttgccc tgtaaacgaa aaaaccacct ggggaggtgg

9001 tttgatcgaa ggttaagtca gttggggaac tgcttaaccg tggtaactgg atttcgcaga

9061 gcacagcaac caaatctgtc cttccagtgt agccggactt tggcgcacac ttcaagagca

9121 accgcgtgtt tagctaaaca aatcctctgc gaactcccag ttaccaatgg ctgctgccag

9181 tggcgtttta ccgtgctttt ccgggttgga ctcaagtgaa cagttaccgg ataaggcgca

9241 gcagtcgggc tgaacgggga gttcttgctt acagcccagc ttggagcgaa cgacctacac

9301 cgagccgaga taccagtgtg tgagctatga gaaagcgcca cacttcccgt aagggagaaa

9361 ggcggaacag gtatccggta aacggcaggg tcggaacagg agagcgcaag agggagcgac

9421 ccgccggaaa cggtggggat ctttaagtcc tgtcgggttt cgcccgtact gtcagattca

9481 tggttgagcc tcacggctcc cacagatgca cccggaaaagc gtctgtttat gtgaactctg

9541 gcaggagggc ggagcctatg gaaaaacgcc accggcgcgg ccctgctgtt ttgcctcaca

9601 tgttagtccc ctgcttatcc acggaatctg tgggtaactt tgtatgtgtc cgcagcgccc

9661 gccgcagtct cacgcccgga gcgtagcgac cgagtgagct agcta

//

Claims (22)

Support bacterial cell comprising a conjugative plasmid, the plasmid encoding a methyltransferase enzyme. Support bacterial cell according to claim 1 in which the support bacterial cell is chosen from a Bacteriodes , Escherichia, Clostridium, Caulobacter , Lactobacillus, Pseudomonas, Bifidobacterium , Salmonella, Listeria, Proteus or Streptococcus cell. Support bacterial cell according to claim 1 or 2, wherein the plasmid comprises an OriT and optionally one or more or all of the tra genes necessary for bacterial conjugation. A method of delivering DNA from a supporting bacterial cell to a target bacterium or an archaeal cell by conjugation, the method comprising bringing the target bacterial species into contact with the supporting bacterial cell according to any one of claims 1- 3, wherein the target bacteria comprises a restriction modification system that cleaves DNA that is unmethylated at a target methylation site of the methyltransferase enzyme. A bacteriophage encoding a methyltransferase enzyme, optionally wherein the methyltransferase enzyme protects against a restriction modification system found in a target bacteria that is pathogenic in humans or animals, and/or in a target bacteria that is associated with biofouling or to microbial corrosion. Mixture of at least 2 bacteriophages of claim 5, each bacteriophage encoding a different methyltransferase enzyme. Bacteriophage or mixture according to claim 5 or 6 respectively, wherein the bacteriophage is of a genus selected from Dhakavirus , Gaprivervirus , Gelderlandvirus , Jiaodavirus , Karamvirus , Krischvirus , Moonvirus , Mosigvirus , Schizotequatrovirus , Slopekvirus and Tequatrovirus . Each phage mentioned here may be an Enterobacteriaceae phage, an E coli phage, a Myoviridae phage, a Tevenvirinae phage, a Tequatrovirus phage, a Caudovirales phage, adeno-associated viruses (AAV), viruses herpes simplex, retroviruses or lentiviruses or may be a Klebsiella phage (e.g. Klebsiella phage PMBT1, Klebsiella phage PKO111, Klebsiella phage phi KpNIH-6, Klebsiella Miro phage, Klebsiella phage vB_KpnM_Kp_V477, Klebsiella phage KP27, Klebsiella phage KP15, Klebsiella phage KP1 or Klebsiella phage JD18), an Acinetobacter phage (e.g. Acinetobacter virus 133), an Aeromonas phage (e.g. Aeromonas virus 65 or of Aeromonas Aeh1), an Escherichia phage (e.g. Escherichia virus RB16, Escherichia virus RB32 or Escherichia virus RB43) or a Pseudomonas phage (e.g. Pseudomonas virus 42) or perhaps a Tevenvirinae phage, e.g. a phage chosen from Table D. A method of delivering bacteriophage DNA to a target bacteria, the method comprising contacting the target bacterial species with a bacteriophage encoding a methyltransferase enzyme, wherein the target bacteria comprises a restriction modification system that cleaves the DNA that is unmethylated at a target site for methylation of the enzyme methyltransferase. Process for methylation of bacteriophage DNA or plasmid or phagemid DNA, the process comprising:
introducing into a production bacterium (i) a polynucleotide encoding a plurality of methyltransferases, or (ii) polynucleotides which together encode a plurality of methyltransferases,
the production, thereby, of a modified production bacterium expressing at least 2 introduced methyltransferases;
infecting the engineered production bacterium with a bacteriophage particle comprising bacteriophage DNA or phagemid DNA or plasmid DNA, thereby methylating said bacteriophage DNA or phagemid DNA or plasmid DNA; And
producing a bacteriophage particle comprising bacteriophage DNA or phagemid DNA or plasmid DNA having a modified methylation pattern, optionally purifying the bacteriophage particle comprising bacteriophage DNA bacteriophage or phagemid DNA or plasmid DNA. A method according to claim 9 wherein the produced bacteriophage DNA or plasmid DNA or phagemid DNA encodes a methyltransferase. A method, supporting bacterial cell, bacteriophage, mixture, plasmid or phagemid according to or disclosed in any one of claims 1 to 10, in which
(i) the MTase is a type I, II or III MTase; Or
(ii) The MTase is selected from Eco0015, EcoK1, EcoAI, M.EcoAO83I, M.EcoMII, M.EcoMIII, M1 M2 Eco31I and M.Eco1167; Or
(iii) The methylated MTase within a recognition sequence chosen from
G A GNNNNNNNG T CA
GGANNNNNNNNATGC
AACNNNNNNGTGC
RT A CNNNNG T G
AAAGNNNNGTT
GG T C TC
CCANNNNNNCTTC
YTC A NNNNNNGTTY ; Or
(iv) the phage, phagemid or plasmid, for example the DNA of the conjugation plasmid of the supporting bacterial cell, codes for several MTases, for example comprising a type I MTase and a type II MTase, or an MTase type I and type III, or a type II and III MTase, or all type I, II and III MTases,
or wherein the producing bacteria encodes multiple MTases that are not native to the producing bacteria, such as a type I and type II MTase, or a type I and type III MTase, or an MTase type II and III, or all type I, II and III MTases. A method, supporting bacterial cell, bacteriophage, phagemid or plasmid mixture according to or as disclosed in any of claims 1 to 11, wherein the supporting bacterial cell plasmid, bacteriophage or phagemid comprises a bacteria-modifying CRISPR array targets to modify a target sequence in the target bacteria. A method, supporting bacterial cell, mixture, bacteriophage, phagemid or plasmid according to claim 12, wherein the plasmid, bacteriophage or phagemid further encodes:
(i) a cas endonuclease, and/or
(ii) a tracrRNA sequence or a DNA sequence expressing a tracrRNA sequence. A pharmaceutical composition comprising a carrier bacterial cell or bacteriophage, mixture, phagemid or plasmid according to or disclosed in any of claims 1 to 13 in combination with a pharmaceutically acceptable carrier or excipient. Method, bacterial cell bacteriophage carrier, mixture, phagemid or plasmid according to or disclosed in any one of claims 4 to 8 and 11 to 14 when dependent on claims 4 to 8, wherein the target bacteria is pathogenic in human or animal. Method for preventing or treating a disease caused by a pathogenic target bacteria in humans or animals, the method comprising the provision of a bacteriophage, a mixture, a phagemid, a plasmid or a bacterial carrier cell according to or disclosed in any one of claims 1 to 13 or a pharmaceutical composition according to claim 14 to a human or animal in need thereof. Bacteriophage, mixture, phagemid, plasmid or supporting bacterial cell according to or disclosed in claims 1 to 13 or pharmaceutical composition according to claim 14, intended for use in a method of preventing or treating a disease caused by a pathogenic target bacterium in humans or animals, the method comprising providing the bacteriophage, the phagemid or the supporting bacterial cell or the pharmaceutical composition to the human or animal in need thereof. A method according to claim 16 or a bacteriophage, phagemid, mixture, plasmid or supporting bacterial cell for use according to claim 17, wherein the pathogenic bacteria is selected from all the bacteria listed in Table 1 of WO 2017/211753A1, which list is specifically incorporated herein by reference, or may be Salmonella, Clostridium, Bifidobacterium, Lactobacillus, Escherichia, Pseudomonas, Caulobacter, Listeria, Proteus and Streptococcus. A method of destroying a target bacterium or of targeted modification of the genome of a target bacterium, the method comprising providing a bacteriophage, phagemid, plasmid or a supporting bacterial cell according to or disclosed in claims 1 to 13 , or a pharmaceutical composition according to claim 14, to the target bacteria, the target bacteria being included in a natural population and associated with microbial corrosion or biological fouling of a substrate or fluid in an industrial or domestic system. Method for destroying a target bacterium or for targeted modification of the genome of a target bacterium according to claim 19, the target bacterium being chosen from a Bacillus (e.g. thuringiensis), an E. coli, a Salmonella, a Listeria, a Clostridium (e.g. botulinum), a sulfate-reducing bacteria, e.g. a Desulfovibrio, a Lactobacillus, a Legionella, a Streptococcus, a Lactococcus or a Listeria. A method of targeted modification of a target bacterial species or strain, the method comprising bringing a target bacteria into contact with a/a support bacterial cell, plasmid, bacteriophage, mixture or phagemid according to claim 12, so that the sequence target of the target bacterial species is modified by CRISPR-mediated DNA modification. Method or method of use according to any one of claims 16-18 and 21, wherein the target bacteria is included in the microbiome of a human or animal.