Modifying bacteriophage

Abstract

A method for producing one or more hybrid bacteriophage host range determinant (HRD) sequences, which comprises: (1) identifying at least two DNA sequences, each encoding an HRD in a series of regions in the DNA sequence, wherein the HRDs are different from one another, (2) incorporating each region into a vector in which each region is flanked by a recognition site of a restriction enzyme capable of cutting DNA at a specific cleavage site outside of the recognition sequence, so that the cleavage site of the restriction enzyme is situated at the boundary of each region, wherein the cleavage site sequences of the regions from an individual series are different from one another and wherein the cleavage site sequences at the boundaries of corresponding regions from different series are the same; (3) treating the vectors with a restriction enzyme capable of cutting DNA at a specific cleavage site outside of the recognition sequence so as to generate a mixture of the regions; and (4) treating the mixture of the regions with a ligase to ligate them to form an array of DNA sequences encoding an array of hybrid HRDs.

Claims

1. A method for producing one or more hybrid bacteriophage host range determinant (HRD) sequences, which comprises: (1) identifying at least two DNA sequences, each encoding an HRD in a series of regions in the DNA sequence, wherein the HRDs are different from one another, (2) incorporating each region into a vector in which each region is flanked by a recognition site of a restriction enzyme capable of cutting DNA at a specific cleavage site outside of the recognition sequence, so that the cleavage site of the restriction enzyme is situated at the boundary of each region, wherein the cleavage site sequences of the regions from an individual series are different from one another and wherein the cleavage site sequences at the boundaries of corresponding regions from different series are the same; (3) treating the vectors with a restriction enzyme capable of cutting DNA at a specific cleavage site outside of the recognition sequence so as to generate a mixture of the regions; and (4) treating the mixture of the regions with a ligase to ligate them to form an array of DNA sequences encoding an array of hybrid HRDs.

2. The method according to claim 1 wherein the restriction enzyme is selected from a Type IIB restriction enzyme and a Type IIS restriction enzyme.

3. The method according to claim 1, wherein steps (3) and (4) are carried out in a single reaction.

4. The method according to claim 1, wherein the restriction enzyme recognition sites are added to each region.

5. The method according to claim 1, wherein the regions are amplified or synthesised prior to incorporation into the vectors.

6. The method according to claim 1, wherein the cleavage site sequence of at least one of the regions is formed by changing the nucleotide base sequence of the region without changing the amino acid sequence encoded by the region.

7. The method according to claim 1, which further comprises (5) incorporating each hybrid HRD from the array of hybrid HRDs into a delivery vector to form an array of delivery vectors.

8. The method according to claim 7, wherein: (a) the array of delivery vectors is contacted with first host cells so as to introduce each delivery vector into a first host cell to form an array of transformed first host cells; (b) the array of transferred first host cells is infected with a target phage; (c) phage replication and recombination are effected; (d) recombinant phage are screened; and (e) recombinant phage bearing hybrid HRDs are selected.

9. The method according to claim 8, wherein steps (d) and (e) comprise propagating recombinant phage on a second host cell which is a host for phage bearing a hybrid HRD and not a host for the target phage.

10. The method according to claim 9, further comprising the steps: (f) the selected recombinant phage bearing hybrid HRDs are contacted with the first host cells so as to infect the first host cells; (g) phage replication is effected; and (h) recombinant phage bearing hybrid HRDs capable of infecting the first host cell and the second host cell are selected.

11. The method according to claim 1, wherein the at least two DNA sequences are identified by HRD nucleotide sequence alignment, HRD amino acid sequence alignment or HRD protein structure alignment.

12. The method according to claim 1, wherein the HRDs comprise tail fibre proteins, or tail fibre proteins wherein each tail fibre protein comprises a receptor binding region for binding to the target bacteria and a region linking the receptor binding region to the body of the bacteriophage.

13. The method according to claim 12, wherein the receptor binding region is a C-terminal receptor binding region and the region linking the C-terminal receptor binding region to the body of the bacteriophage is an N-terminal region.

14. The method according to claim 13, wherein the N-terminal region comprises amino acids 1 to 628 of the tail fibre protein and the C-terminal region comprises amino acids 629 to 964 of the tail fibre protein, based on the amino acid sequence of bacteriophage Phi33.

15. The method according to claim 2, wherein the Type IIS restriction enzyme is selected from BsaI, BpiI, BcoDI, BbvI, BbsI, BsmAI, BsmFI, FokI, SfaNI, BfuAI, BsmBI, BspMI, BtgZI, and Esp3I, or an isoschizomer thereof, or wherein the Type IIS restriction enzyme is selected from EarI, BspQI and SapI, or an isoschizomer thereof, or wherein the Type IIS restriction enzyme is HgaI, or an isoschizomer thereof, and/or wherein the Type IIB restriction enzyme is selected from AlfI, AloI, BaeI, BsgI, BplI, BsaXI, CspCI, FalI, PpiI and PsrI, or an isoschizomer thereof.

16. The method according to claim 8 wherein the recombinant phage bearing hybrid HRDs are provided with a gene encoding a protein which is toxic to a target bacterium.

17. The method according to claim 16 wherein the gene encodes an /-type small acid-soluble spore protein (SASP), a SASP-C, or a SASP-C from Bacillus megaterium.

18. The method according to claim 8, wherein step (e) comprises selecting a recombinant phage bearing hybrid HRDs which confer a host range which is broader than a host range of the target phage.

19. The method according to claim 8, wherein step (e) comprises selecting a recombinant phage bearing hybrid HRDs which confer a host range comprising the host ranges of the HRD sequences encoded by the at least two DNA sequences.

20. The method according to claim 8, wherein step (e) comprises selecting a recombinant phage bearing hybrid HRDs having a broad host range as defined by more than 50% of a collection of at least 35 and preferably more than 50 clinical isolates, from a plurality of different infection sites and including range of antibiotic resistance phenotypes.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) FIG. 1. Infectivity of phage PTP238, Phi33, PTP47 and PTP92. Infectivity assessed by inoculating phage on to agar seeded with P. aeruginosa bacteria (strains listed in figure), and assessing the growth of the bacterial lawn in the region of the inoculum. Growth inhibition is marked +, indicating that the phage infects the host strain. Strains which are not infected by the phage are marked.

(2) FIGS. 2A-2B. Summary of HRD modules 1A-1F and 2A-2F. The sequences of the cleavage sites (cs) and their abbreviations are shown, along with the sequences of the module fragments that could be generated by synthesis, and the plasmids that could be constructed by cloning these fragments into a derivative of pUC57 from which the native BsaI restriction site has been silently removed.

(3) FIG. 3. Schematic summarising the method for creating recombinant bacteriophage carrying chimeric HRD protein, following construction of a chimeric HRD library.

(4) FIGS. 4A-4B. Schematic summary of the method of the present invention for the creation of a phage carrying in chimeric HRD proteins. (A) For each phage, set of six plasmids are created to contain the coding sequence for six regions (labelled A-F) of the phage's HRD protein (HRD1 and 2). Each region is delineated by a cleavage site (labelled cs1-7): specific for the junction (or termini) of each region. When cloned in the plasmid: the cleavage sites are flanked by recognition sites for a Type IIS restriction enzyme (BsaI in this instance). For the example shown there are two phage HRD sequences, HRD1 and HRD2 from phage 1 and 2 respectively, and thus 12 plasmids created in total. (B) The 12 plasmids, together with a plasmid delivery vector, are digested with a BsaI and ligated together in a one-pot reaction to form plasmids which carry the coding sequence for chimeric HRD proteins which are randomly mixed in the six regions A-F, flanked homology arms (HA) for recombination with the target have. A delivery vector carrying a mixture of DNA sequences is shown as an example.

(5) FIGS. 5A-5E. CLUSTAL Omega multiple sequence alignment of the tail fibre proteins from Phage SPM-l, F8, PBl, C36, LBL3, Phi33, LMA2, KPP12, JG024, PTP92, NH-4, 14-1, PTP47, SN. Sequence divergent C-terminal region shaded in grey, sequence conserved N-terminal region unshaded.sss

(6) FIGS. 6A-6D. Needle (EMBOSS) pairwise alignment of the tail fibre genes from Phage PTP92 and PTP47. The sequences used as cleavage sites, when cloned into plasmid vectors with flanking restriction enzyme recognition sites, are shaded in black. Nucleotides shown in bold and underlined are changed to corresponding nucleotide sequence in the second aligned sequence upon synthesis and cloning. The translated protein sequence is preserved upon nucleotide sequence change.

(7) FIGS. 7A-7B. CLUSTAL Omega multiple sequence alignment of the tail fibre proteins from Phage PTP92 and PTP47. The residues which correspond to the boundaries of the DNA sequence regions cloned in the plasmid libraries are shown shaded in black.

(8) FIG. 8. Sequence of the tail fibre protein from PTP238. Sequences PTP92 are shown in bold and sequences from PTP47 are underlined. The remainder of the sequences is from Phi33, The boundaries of the sequence regions are marked by residues which are shaded black.

(9) FIGS. 9A-9C. A schematic diagram showing one way of isolating recombinant bacteriophage that have acquired the host range of two or more donor bacteriophage.

DETAILED DESCRIPTION OF INVENTION

(10) Summary of a method for the genetic modification of a bacteriophage such that it carries chimeric tail fibre variants that confer a desired altered host range.

(11) As an example only, it is shown here how the amino acid sequences of phage host range determinants, such as those from PTP92 and PTP47 may be aligned, and how the regions of conservation and variation thus identified may be used to divide one of the HRD-encoding genes (such as that from PTP92) into several modules. The sequence alignment can then be used to divide any other HRD-encoding genes under consideration (in this example, PTP47) into positionally-corresponding modules.

(12) It is shown how the HRD modules thus defined from PTP92 and PTP47 may each be flanked by TypeIIS (BsaI) restriction sites and suitable cleavage sites (cs). It is then shown how a plasmid library consisting of every possible combination of PTP92 and PTP47 HRD modules may be constructed by Golden Gate assembly, to form a plasmid library of chimeric HRDs.

(13) There are several ways in which phages carrying non-native genetic DNA sequences can be constructed and the following is an example of such methods. One way in which genes can be removed and added to the phage genome is by using homologous recombination. Here, it is shown, as an example, how the chimeric HRDs can be cloned in between a Phi33 sequence located immediately upstream of the corresponding identified variable region of the Phi33 HRD, and another Phi33 sequence located immediately downstream of the corresponding Phi33 HRD. These Phi33 sequences can act as regions of homology for homologous recombination with Phi33 bacteriophage. The resulting plasmid library of chimeric HRDs cloned in between Phi33 regions of homology can then be transferred to a P. aeruginosa strain that is a host for Phi33, PTP92 and PTP47.

(14) To isolate Phi33 derivatives which have undergone recombination with the chimeric HRDs library, a Phi33 lysate may be made on a mixed culture of P. aeruginosa, each cell of which carries a representative of the chimeric HRDs library, and where each representative of the chimeric HRDs library is present in the mixed culture. The resulting lysate may then be propagated on a P. aeruginosa strain that is a host for PTP92, but not Phi33 or PTP47, to isolate recombinant phage that have acquired the plaquing ability of PTP92. A second round of phage propagation may be carried on the same PTP92 host P. aeruginosa strain to enrich the resulting lysate for the desired recombinant phage that have acquired the plaquing ability of PTP92. This second lysate may then be plagued on a P. aeruginosa strain that is a host for PTP47, but not Phi33 or PTP47, as a means of isolating recombinant phage that have acquired the plaquing ability of PTP47. After plaque purification on the same PTP47 host P. aeruginosa strain, individual plaques may then be tested for plaquing on the P. aeruginosa strain that is a host for PTP92, but not PTP47 or Phi33. Phages that plaque on both the PTP47 discriminatory host and the PTP92 discriminatory host have acquired the host range of both PTP47 and PTP92, and are likely to carry genes encoding chimeric HRD proteins. The HRD region from phage thus identified may be amplified by PCR and sequenced, or alternatively genomic DNA from the phage may be isolated and submitted to whole genome sequencing, to identify the sequence of the chimeric HRD that confers the desired dual host range of PTP92 and PTP47 upon the recombinant Phi33 derivatives.

Experimental Procedures

(15) PCR reactions to generate DNA for cloning purposes may be carried out using Herculase II Fusion DNA polymerase (Agilent Technologies), depending upon the melting temperatures (T.sub.m) of the primers, according to manufacturer's instructions. Alternatively, DNA for cloning may be obtained via custom DNA synthesis, for example by GenScript or DNA 2.0. PCR reactions for screening purposes may be carried out using Taq DNA polymerase (NEB), depending upon the T.sub.m of the primers, according to manufacturer's instructions. Unless otherwise stated, general molecular biology techniques, such as restriction enzyme digestion, agarose gel electrophoresis, T4 DNA ligase-dependent ligations, competent cell preparation and transformation may be based upon methods described in Sambrook et al., (1989). Enzymes may be purchased from New England Biolabs or Thermo Scientific. DNA may be purified from enzyme reactions and prepared from cells using Qiagen DNA purification kits. Plasmids may be transferred from E. coli strains to P. aeruginosa strains by conjugation, mediated by the conjugation helper strain E. coli HB101 (pRK2013). A chromogenic substrate for -galactosidase, S-gal, that upon digestion by -galactosidase forms a black precipitate when chelated with ferric iron, may be purchased from Sigma (S9811).

(16) Primers may be obtained from Sigma Life Science. Where primers include recognition sequences for restriction enzymes, additional 2-6 nucleotides may be added at the 5 end to ensure digestion of the PCR-amplified DNA.

(17) Standard clonings may be achieved by ligating DNAs overnight with T4 DNA ligase and then transforming them into E. coli cloning strains, such as DH5a or TOP10, with isolation on selective medium, as described elsewhere (Sambrook et al., 1989). Clonings involving TypeIIS restriction enzymes may be achieved by incubating the DNAs simultaneously with T4 DNA ligase and with the relevant TypeIIS restriction enzyme, in T4 DNA ligase buffer, using a thermal cycler programmed as follows:

(18) TABLE-US-00001 Temperature Time Number of cycles 37 C. 2 hours 1 37 C. 2 minutes 50 16 C. 3 minutes 50 C. 5 minutes 1 80 C. 5 minutes 1 16 C. Hold 1

(19) An E. coli/P. aeruginosa broad host range vector, such as pSM1484A, may be used to transfer genetic material between E. coli and P. aeruginosa. This type of vector is otherwise known as a delivery vector. Plasmid pSM1484A is a previously engineered construct carrying a broad-host-range, low copy origin of replication from a P. aeruginosa plasmid, an E. coli-specific high-copy origin of replication from plasmid pUC19, the oriT origin of transfer from plasmid RP4, a tetracycline resistance marker, and sequence modified from phage Phi33. The latter sequence comprises the conserved region of Phi33's HRD, silently mutated to suppress an intrinsic BsaI site, followed by a CTCGtGAGACC (SEQ ID NO: 1) BsaI site containing cs1 (CTCG), a lacZa reporter gene, a second BsaI site GGTCTCaAATG (SEQ ID NO: 2) containing cs7 (AATG), and finally sequence from Phi33 corresponding to sequence downstream of the HRD gene's stop codon in the native genome.

(20) Detection of Phi33-like phage (PB1-like phage family) conserved N-terminal tail fibre regions by PCR 1. Primers for the detection of Phi33-like phage-like tail fibre genes in experimental phage samples may be designed as follows:

(21) The DNA sequences of the tail fibre genes from all sequenced Phi33-like phage (including Phi33, PB1, NH-4, 14-1, LMA2, KPP12, JG024, F8, SPM-1, LBL3, PTP47, C36, PTP92 and SN) may be aligned using Clustal Omega, which is available on the EBI website, and the approximately 2 kb-long highly conserved region mapping to the gene's 5 sequence may be thus identified (positions 31680-33557 in the PB1 genome sequence, Acc. EU716414). Sections of 100% identity among the 11 tail fibre gene sequences may be identified by visual inspection. Three pairs of PCR primers targeting selected absolutely conserved regions, and amplifying PCR products no longer than 1 kb may be chosen as follows: pair B4500 and B4501, defining a 193 bp-long region; pair B4502 and B4503, defining a 774 bp-long region; and pair B4504 and B4505, defining a 365 bp-long region.

(22) Primer B4500 consists of sequence of PB1 phage genome (Acc. EU716414) ranging from position 31680 to 31697. Primer B4501 consists of sequence of PB1 phage genome (Acc. EU716414) ranging from position 31851 to 31872. Primer B4502 consists of sequence of PB1 phage genome (Acc. EU716414) ranging from position 31785 to 31804. Primer B4503 consists of sequence of PB1 phage genome (Acc. EU716414) ranging from position 32541 to 32558. Primer B4504 consists of sequence of PB1 phage genome (Acc. EU716414) ranging from position 32868 to 32888. Primer B4505 consists of sequence of PB1 phage genome (Acc. EU716414) ranging from position 33213 to 33232.

(23) TABLE-US-00002 B4500 (SEQ ID NO: 3) 5-GTGATCACACCCGAACTG-3 B4501 (SEQ ID NO: 4) 5-CGATGAAGAAGAGTTGGTTTTG-3 B4502 (SEQ ID NO: 5) 5-ACGCCGGACTACGAAATCAG-3 B4503 (SEQ ID NO: 6) 5-TCCGGAGACGTTGATGGT-3 B4504 (SEQ ID NO: 7) 5-CCTTTCATCGATTTCCACTTC-3 B4505 (SEQ ID NO: 8) 5-TTCGTGGACGCCCAGTCCCA-3

(24) 2. Phi33-like tail fibre genes may be detected in experimental phage samples as follows:

(25) Plaques of isolated phage of environmental origin may be picked from agar plates and added to water and incubated for 30 minutes, making plaque soak outs. The plaque soak outs may be diluted and a portion added to PCR reactions containing one or all of the above primer pairs, and PCR may be performed according to a standard protocol. PCR products may be visualised on a 1.5% agarose gel with ethidium bromide staining, and evaluated for their size. PCR products of the correct size for the primer pair used may be gel-extracted and submitted to an external facility for sequencing. Sequencing results may be compared with the available tail fibre gene sequences in order to confirm the identity of the PCR product.

(26) An example of the construction of chimeric HRDs from two parental HRDs.

(27) Selection of Module Boundaries 1. The amino acid sequences of the parental HRDs may be aligned using ClustalOmega and regions of highest sequence conservation may be so identified (FIGs. 7A-7B). 2. To generate chimeric HRDs comprising six modules, seven 4 nt cleavage sites (cs1 to cs7; FIGS. 2A-2B) may be selected from the most highly conserved regions of the alignment, according to the criteria previously outlined. As an example, i) cs1 (CTCG) forms the 5 boundary of module A (FIGS. 7A-7B; FIGS. 2A-2B) and is identical to that engineered into the acceptor site of the recipient plasmid, the delivery vector pSM1484A, at the junction between the conserved and hyper-variable regions of either HRD's full-length DNA sequence; ii) cs2 corresponds to GGAT, 162 nt downstream of cs1; iii) cs3, introducing a silent point mutation in HRD1, corresponds to GCGG 646 nt downstream of cs2; iv) cs4, introducing a silent point mutation in HRD1, corresponds to GGGA 37 nt downstream of cs3; v) cs5, introducing a silent point mutation in HRD1, corresponds to ACTC 28 nt downstream of cs4; vi) cs6 corresponds to TGGG 56 nt downstream of cs5; and finally, vii) cs7, corresponding to AATG 64 nt downstream of cs6, immediately downstream of the HRD stop codon, is identical to that engineered into the acceptor site of the recipient plasmid, the delivery vector pSM1484A.

(28) Design and Cloning of Module Sequences 1. DNA fragments consisting of the different modules, flanked by suitable cs on either side, and with inverted BsaI restriction sites on either end of the DNA fragment may be generated by custom DNA synthesis. These services are widely available from companies such as Genscript and DNA2.0. The sequences of modules A-F, including flanking BsaI restriction sites and 4 bp cs, for both PTP92 and PTP47, are shown in FIGS. 2A-2B. As an alternative to custom DNA synthesis, another option may be to amplify the modules by PCR, with appropriate primers that incorporate the BsaI restriction sites and 4nt cs, using PTP92 or PTP47 DNA as template for the reactions. 2. Custom synthesised DNA modules may be subcloned into a suitable E. coli plasmid vector that lacks intrinsic BsaI restriction sites, and which uses an alternative selectable marker to that being used on the delivery vector. If pSM1484A is used as the delivery vector, the selectable marker for the module vectors must be something other than tetracycline resistance. A suitable vector could be a derivative of pUC57 from which the intrinsic BsaI restriction site has been silently removed (pUC57(BsaI)). A library of clones in pUC57(BsaI), each carrying a different module, may be thus generated. An example of the DNA sequences cloned in such a library is shown in FIGS. 2A-2B.

(29) Construction of a Plasmid Library Containing Chimeric HRD, and Subsequent Transfer to E. coli 1. The acceptor plasmid, pSM1484A, and the module plasmids, pSMG1 to pSMG12 (FIGS. 2A-2B) may be prepared using standard methods, then quantified using a nanodrop, or by gel electrophoresis and comparison with a known standard. 2. The quantities of pSM1484A and pSMG1 to pSMG12 to use in each digestion/ligation reaction should be calculated such that the delivery vector (pSM1484A) and each of the modules A-F are present in equimolar amounts in the reaction. As an example, the amount of pSM1484A may be fixed at 200 ng in a 20 l reaction. The quantity of module plasmid (Q) (e.g. pSMG1, pSMG2, pSMG3, pSMG4, pSMG5, pSMG6, pSMG7, pSMG8, pSMG9, pSMG10, pSMG11 or pSMG12) required may be calculated according to the equation:

(30) $Q=\frac{\frac{M}{A}P}{N}$

(31) Where

(32) M is the sequence length of the module plasmid (or DNA fragment length, if linear DNA molecules are being used instead of plasmids) in base pairs

(33) A is the sequence length of the delivery vector in base pairs

(34) Q is the quantity of module plasmid (or linear DNA fragment, if these are being used instead of plasmids) required in ng

(35) P is the amount of delivery vector being used in the reaction in ng

(36) N is the number of alternatives available for each module

(37) Taking pSMG1 as an example,

(38) M=2894 bp

(39) A=14039 bp

(40) P may be fixed at 200 ng, as described above

(41) N=2 (in this example, there are two alternatives for the module, i.e. module 1A and module 2A, originating from the HRD of PTP92 and PTP47 respectively).

(42) Quantity of pSMG1 required per 200 ng of delivery vector,

(43) $\begin{array}{c}\mathrm{pSM}1484A=\frac{\frac{2894}{14039}200}{2}\\ =20\mathrm{ng}\end{array}$ 3. The delivery vector and all of the module plasmids may then be mixed according to the calculated values in a single, 20 l one-tube reaction that also contains T4 DNA ligase buffer, BsaI (20 Units), and T4 DNA ligase (40 Units). The assembled reaction may be placed in a thermal cycler for incubation at alternating temperatures as follows: 37 C. for 2 hours, then 50 (37 C., 2 minutes; 16 C., 3 minutes), then 50 C., 5 minutes, then 80 C., 10 minutes, and finally hold at 16 C. 4. The reaction may then be isopropanol-precipitated, resuspended in water and used to transform E. coli DH10B, according to standard protocols. 5. Successful clones derived from pSM1484A may be isolated by plating transformants onto medium containing tetracycline. Additionally, the plates may also contain a suitable substrate to visualise -galactosidase activity, such as the chromogenic substrate S-gal. If required, clones carrying pSM1484A derivatives in which the lacZa reporter gene has been replaced by an insert between the vector BsaI restriction sites will appear white on S-gal plates, and thus may be distinguished from clones carrying the parental pSM1484A plasmids, which retain the lacZa reporter, and so will appear black on S-gal plates. 6. E. coli transformants containing the plasmid library may be harvested by aseptically scraping white colonies off the transformation plates, pooling them and resuspending in 10 ml LB. Ideally, 10.sup.5-10.sup.6 transformant colonies should be pooled. The pooled library mixture may be either used directly, or may be stored in 25% glycerol (final concentration) at 20 C. for future use.

(44) Generation of Phage Carrying Chimeric HRD, Via Recombination with the Plasmid Library 1. The pool of E. coli transformants harbouring the plasmid library may be used directly, or the glycerol stocks can be used to inoculate a fresh culture, for further work. 2. The plasmid library may be transferred from the pool of E. coli transformants, to P. aeruginosa strain 1868, which is a host for all three phage (Phi33, PTP47 and PTP92), by conjugation (FIGS. 9A-9C). Transformants may be selected on the basis of acquisition of tetracycline resistance. 3. The P. aeruginosa transformants containing the plasmid library may be harvested by aseptically scraping colonies off the transformation plates and pooling them and resuspending in 10 ml LB. Ideally, 10.sup.5-10.sup.6 transformant colonies should be pooled. The pooled library mixture may either be used directly, or may be stored in 25% glycerol (final concentration) at 20 C. for future use. 4. The pool of P. aeruginosa transformants harbouring the plasmid library may then be used as a host on which a Phi33 phage lysate can be made (FIGS. 9A-9C). The resulting lysate (lysate ; FIGS. 9A-9C) should contain recombinant phage that have acquired chimeric HRD by homologous recombination with the plasmid library. 5. Lysate , the Phi33 recombinant library, may then be plagued on Pseudomonas strain 2726, that is a host for PTP92, but not PTP47 or Phi33, and a new lysate made (lysate ), to isolate recombinant phage that have acquired the PTP92 host range associated with acquisition of elements of HRD1 (FIGS. 9A-9C). . Lysate 13 can then be re-plaqued on Pseudomonas strain 2726 and a second new lysate made (lysate 7), to enrich the phage library for candidates that have the PTP92 host range (FIGS. 9A-9C). 7. To isolate recombinant phage that have also acquired the PTP47 host range, lysate may be plagued on Pseudomonas strain 2944 that is a host for PTP47, but not PTP92 or Phi33 (FIGS. 9A-9C). 8. Individual plaques resulting from step 7 may be plaque purified on Pseudomonas strain 2944, and re-tested on Pseudomonas strain 2726, to confirm acquisition of the host ranges of both PTP92 and PTP47 (FIGS. 9A-9C). 9. The HRD region from the resulting purified phage that have the desired host range may be amplified by PCR using primers that bind to Phi33 genomic sequences located upstream and downstream of the regions of homology that were cloned into pSM1484A. The PCR product may then be sequenced to determine the sequence of the chimeric HRD (FIGS. 9A-9C). 10. One such phage that acquired the ability to plaque on both the PTP92 host (2726) and the PTP47 host (2944) was PTP238. Sequence analysis of the HRD of PTP238 revealed that it carried a chimeric HRD with the module organisation A2-B1-C2-D1-E1-F1, where modules designated 1 originated from PTP92, and modules designated 2 originated from PTP47. The amino acid sequence of the HRD from PTP238 is shown in FIG. 8.

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