MODIFIED BACTERIOPHAGE

Abstract

A modified bacteriophage capable of infecting a plurality of different target bacteria, which bacteriophage includes a toxin gene encoding a toxin protein which is toxic to the target bacteria; wherein the bacteriophage is lytic; and wherein the bacteriophage expresses host range determinant proteins which have a plurality of bacterial host specificities.

Claims

1. A modified bacteriophage capable of infecting a plurality of different target bacteria, which bacteriophage includes a toxin gene encoding a toxin protein which is toxic to the target bacteria; wherein the bacteriophage is lytic; and wherein the bacteriophage expresses host range determinant proteins which have a plurality of bacterial host specificities.

2-45. (canceled)

46. A modified bacteriophage capable of infecting a plurality of different target bacteria, which bacteriophage comprises a variant of bacteriophage Phi33 which has been modified to comprise an / small acid-soluble spore protein (SASP) gene encoding a SASP gene which is expressed under the control of a constitutive promoter, and further wherein the bacteriophage is lytic.

47. A modified bacteriophage according to claim 46, wherein the tail fiber protein comprises a receptor binding region that binds to the target bacterium and a region linking the receptor binding region to the body of the bacteriophage.

48. A modified bacteriophage according to claim 46, wherein the constitutive promoter is selected from pdhA, rspB, pgi, fda, and lasB.

49. A modified bacteriophage according to claim 46, wherein the constitutive promoter is fda.

50. A modified bacteriophage according to claim 46, wherein the SASP is SASP-C.

51. A modified bacteriophage according to claim 50, wherein the SASP-C is from Bacillus megaterium.

52. A modified bacteriophage according to claim 46, wherein the target bacteria includes Pseudomonas.

53. A modified bacteriophage according to claim 46, wherein the target bacteria includes Pseudomonas aeruginosa.

54. A composition comprising a modified bacteriophage according to claim 46, and a pharmaceutically acceptable carrier.

55. A composition comprising a modified lytic bacteriophage according to claim 46 in admixture with at least one other modified lytic bacteriophage which is capable of infecting target bacteria, which also includes a SASP gene encoding a SASP which is toxic to a target bacteria.

56. A composition according to claim 55, which comprises at least two of said modified lytic bacteriophages, wherein at least two of which have different host specificities.

57. A modified bacteriophage according to claim 46, which comprises a SASP-C gene comprised of P. aeruginosa optimized codons, which gene is operably linked to the fda promoter.

58. A modified bacteriophage according to claim 46, which consists of a variant of bacteriophage Phi33 wherein the modifications consist of the incorporation of a SASP-C gene which is comprised of P. aeruginosa optimized codons, and which gene is operably linked to the fda promoter, and further wherein the bacteriophage is lytic.

59. A composition according to claim 54 which is formulated for pharmaceutical use, for topical use, or for delivery to the respiratory tract.

60. A composition according to claim 58 which is formulated for pharmaceutical use, for topical use, or for delivery to the respiratory tract.

61. A method of killing target bacteria comprising contacting said target bacteria with a bactericidally effective amount of at least one modified bacteriophage according to claim 46, which is toxic to the target bacteria.

62. A method of killing target bacteria comprising contacting said target bacteria with a bactericidally effective amount of at least one modified bacteriophage according to claim 58, which is toxic to the target bacteria.

63. The method of claim 61, which is effected in vitro. or in vivo, by administering a bactericidally effective amount of said modified bacteriophage to a subject infected by said target bacterium.

64. The method of claim 62, which is effected in vitro. or in vivo, by administering a bactericidally effective amount of said modified bacteriophage to a subject infected by said target bacterium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0047] This invention will now be described in more detail, by way of example only, and with reference to the accompanying drawings, in which:

[0048] FIGS. 1A and 1B are a schematic diagram showing construction of a plasmid containing lacZM15

[0049] FIGS. 2A, 2B, 2C, and 2D are a schematic diagram showing construction of plasmids with replaced tail fibre sections;

[0050] FIGS. 3A, 3B, 3C, and 3D are a schematic diagram showing construction of phage with hybrid tail fibre genes, which may be subsequently modified to introduce SASP-C according to the invention;

[0051] FIGS. 4A, 4B, 4C, and 4D are a schematic diagram showing construction of phage with further hybrid tail fibre genes, which may be subsequently modified to introduce SASP-C according to the invention;

[0052] FIGS. 5A and 5B are a schematic diagram showing construction of bacteriophage with hybrid tail fibre genes, in which the lacZ marker has been removed;

[0053] FIGS. 6A, 6B, 6C, and 6D are a schematic diagram showing construction of plasmids in which SASP-C is introduced into a suitable Phi33 insertion site;

[0054] FIGS. 7A and 7B are a schematic diagram showing production of further bacteriophage according to the invention;

[0055] FIGS. 8A, 8B, 8C, and 8D are a schematic diagram showing construction of plasmids in which SASP-C codon optimised for expression in P. aeruginosa is introduced into a suitable Phi33 insertion site;

[0056] FIG. 9 shows the sequence of the SASP-C gene from Bacillus megaterium strain KM (ATCC 13632), which has been codon optimised for expression in P. aeruginosa; and contains SEQ ID NO: 59;

[0057] FIGS. 10A, 10B, 10C, and 10D are a schematic diagram showing production of bacteriophage in which SASP-C which has been codon optimised for expression in P. aeruginosa is introduced into a suitable Phi33 insertion site;

[0058] FIGS. 11A, 11B, 11C, and 11D are a schematic diagram showing production of further bacteriophage according to the invention;

[0059] FIGS. 12A and 12B are a schematic diagram showing production of further bacteriophage according to the invention;

[0060] FIGS. 13A, 13B, 13C, 13D and 13E are a CLUSTAL 2.1 multiple sequence alignment of the tail fibre proteins from Phage SPM-1, F8, PB1, C36, LBL3, Phi33, LMA2, KPP12, JG024, PTP92, NH-4, 14-1, PTP47, SN; and contains SEQ ID NOs: 45-58; and

[0061] FIGS. 14A-14B. 24 hour time-kill curve showing the in vitro efficacy of PT3.9 against P. aeruginosa strains 3503 (FIG. 14A) and ATCC 27853 (FIG. 14B). Cultures were grown in Luria bertani (LB) broth supplemented with 10 mM calcium chloride, 1 mM magnesium sulphate and 1% glucose, at 37 C.

[0062] FIG. 15. In vivo efficacy of PT3.9 in a murine bacteraemia model of infection. Mice were treated IV with vehicle (tris buffered saline containing 1 mM magnesium sulphate, 10 mM calcium chloride and 10% v/v glycerol), PTP284 or PTP387 (2 hours post infection with P. aeruginosa). The viable cell counts in liver tissue at 22 hours post treatment are shown for each animal in each group (group size=6), the geomean for each data set is represented by a horizontal line.

[0063] FIG. 16. In vivo efficacy of PT3.9 in a neutropenic murine pneumonia model of infection. Mice were treated IT (intra-trachea) with placebo vehicle (tris buffered saline containing 1 mM magnesium sulphate, 10 mM calcium chloride and 10% v/v glycerol), PT3.9, PT3.8 or Tobramycin (15 minutes post infection with P. aeruginosa). The viable cell counts in lung tissue at 24 hours post treatment are shown for each animal in each group (group size=6), the geomean for each data set is represented by a horizontal line.

[0064] The product of the invention provides in one aspect a single tail fibre within an individual phage, or a mix of phages where each type of phage has a single, different tail fibre.

[0065] This is a summary of the genetic modification of a lytic bacteriophage to render it non-lytic, such that it carries one of a number of possible tail fibre variants, in addition to SASP-C under the control of a promoter that usually controls expression of the Pseudomonas aeruginosa 30S ribosomal subunit protein S2 gene (rpsB), or SASP-C codon optimised for expression in P. aeruginosa, under the control of a promoter that usually controls expression of the P. aeruginosa fructose-1,6-bisphosphate aldolase gene (fda).

[0066] Genes can be removed and added to the phage genome using homologous recombination. There are several ways in which phages carrying foreign genes and promoters can be constructed and the following is an example of such methods.

[0067] For the construction of a Phi33 derivative it is shown how, using an E. coli/P. aeruginosa broad host range vector, as an example only, the existing tail fibre, or a section of the tail fibre, in the bacteriophage genome may be replaced by an alternative tail fibre or tail fibre section from a different bacteriophage, via homologous recombination. It is also shown as an example only, how additional DNA sequences, such as the SASP-C gene from B. megaterium under the control of a P. aeruginosa rpsB promoter, or the SASP-C gene from B. megaterium, codon optimised for expression in P. aeruginosa, under the control of a P. aeruginosa fda promoter may be added to the bacteriophage genome via homologous recombination.

[0068] A tail fibre gene, or section of a tail fibre gene, from an alternative phage may be cloned between two regions of Phi33 DNA that flank the native tail fibre, or section thereof, along with a lacZ genetic marker, in a broad host range E. coli/P. aeruginosa vector. This plasmid may be introduced into P. aeruginosa, and the resulting strain infected with Phi33. Following harvesting of progeny phage, double recombinants in which the native Phi33 tail fibre or tail fibre section, has been replaced by the new tail fibre or tail fibre section and lacZ, may be isolated by plaquing on a suitable P. aeruginosa (lacZM15+) host strain using medium containing a chromogenic substrate that detects the action of -galactosidase.

[0069] In a subsequent step, the lacZ marker may be removed from the bacteriophage genomes by making versions of the previously described tail fibre region recombination plasmids that do not contain the lacZ marker, introducing the new plasmids into suitable P. aeruginosa host strains and infecting with the previously modified bacteriophage derivatives of Phi33 carrying the corresponding alternative tail fibre gene, or section thereof, along with the lacZ marker. Recombinants that retain the new tail fibre or tail fibre section, but from which lacZ has been removed, may be isolated by plaquing on a suitable P. aeruginosa (lacZM15+) host strain using medium containing a chromogenic substrate that detects the action of -galactosidase.

[0070] In a subsequent step, a similar homologous recombination may be used to introduce the gene for SASP-C, under the control of a P. aeruginosa rpsB promoter, while simultaneously adding an E. coli lacZ reporter gene for the identification of recombinant phage, into Phi33, or any of the previously described Phi33 derivatives, or similar bacteriophage or similar derivatives. A region consisting of SASP-C controlled by the rpsB promoter, and the E. coli lacZ allele, may be cloned between two regions of Phi33 that flank a suitable insertion site, such as the intergenic region located immediately downstream of the Phi33 tail fibre operon, in a broad host range E. coli/P. aeruginosa vector. This plasmid may be transferred to a suitable P. aeruginosa (lacZM15+) strain, and the resulting strain infected by Phi33 or the previously constructed Phi33 derivative (from which the initial lacZ marker has been removed). Progeny phage may be harvested and double recombinants identified by plaquing on P. aeruginosa (lacZM15+), looking for acquisition of the new lacZ reporter on medium containing a chromogenic substrate that detects the action of -galactosidase.

[0071] In an alternative subsequent step, a similar homologous recombination may be used to introduce the gene for SASP-C that has been codon optimised for expression in P. aeruginosa, under the control of a P. aeruginosa fda promoter, while simultaneously adding an E. coli lacZ reporter gene for the identification of recombinant phage, into Phi33, or any of the previously described Phi33 derivatives, or similar bacteriophage or similar derivatives. A region consisting of codon optimised SASP-C controlled by the fda promoter, and the E. coli lacZ allele, may be cloned between two regions of Phi33 that flank a suitable insertion site, such as the intergenic region located immediately downstream of the Phi33 tail fibre operon, in a broad host range E. coli/P. aeruginosa vector. This plasmid may be transferred to a suitable P. aeruginosa (lacZM15+) strain, and the resulting strain infected by Phi33 or the previously constructed Phi33 derivative (from which the initial lacZ marker has been removed). Progeny phage may be harvested and double recombinants identified by plaquing on P. aeruginosa (lacZM15+), looking for acquisition of the new lacZ reporter on medium containing a chromogenic substrate that detects the action of -galactosidase.

[0072] Since these bacteriophage to be modified are lytic (rather than temperate), another requirement for these described steps of bacteriophage construction is the construction of a suitable host P. aeruginosa strain that carries the E. coli lacZM15 gene at a suitable location in the bacterial genome, to complement the lacZ phenotypes of the desired recombinant bacteriophage. As an example, the construction of these P. aeruginosa strains may be achieved via homologous recombination using an E. coli vector that is unable to replicate in P. aeruginosa. The genomic location for insertion of the lacZM15 transgene should be chosen such that no essential genes are affected and no unwanted phenotypes are generated through polar effects on the expression of adjacent genes. As an example, one such location may be immediately downstream of the P. aeruginosa strain PAO1 phoA homologue.

[0073] The E. coli lacZM15 allele may be cloned into an E. coli vector that is unable to replicate in P. aeruginosa, between two regions of P. aeruginosa strain PAO1 genomic DNA that flank the 3 end of phoA. This plasmid may be introduced into P. aeruginosa and isolates having undergone a single homologous recombination to integrate the whole plasmid into the genome selected according to the acquisition of tetracycline (50 g/ml) resistance. Isolates (lacZM15+) which have undergone a second homologous recombination event may then be isolated on medium containing 10% sucrose (utilising the sacB counter-selectable marker present on the plasmid backbone).

[0074] In a subsequent step, a similar homologous recombination may be used to remove the lacZ marker from the previously described, (lacZ+) Phi33 derivatives that have been modified to introduce the gene for SASP-C, under the control of a P. aeruginosa rpsB promoter. A region consisting of SASP-C controlled by the rpsB promoter, may be cloned between two regions of Phi33 that flank the chosen insertion site, in a broad host range E. coli/P. aeruginosa vector. This plasmid may be transferred to a suitable P. aeruginosa (lacZM15+) strain, and the resulting strain infected by the previously described (lacZ+)Phi33 derivatives that have been modified to introduce the gene for SASP-C, under the control of a P. aeruginosa rpsB promoter. Progeny phage may be harvested and double recombinants identified by plaquing on P. aeruginosa (lacZM15+), looking for loss of the lacZ reporter on medium containing a chromogenic substrate that detects the action of -galactosidase.

[0075] In an alternative subsequent step, a similar homologous recombination may be used to remove the lacZ marker from the previously described, (lacZ+) Phi33 derivatives that have been modified to introduce the gene for SASP-C, codon optimised for expression in P. aeruginosa, under the control of a P. aeruginosa fda promoter. A region consisting of SASP-C, codon optimised for expression in P. aeruginosa, controlled by the fda promoter, may be cloned between two regions of Phi33 that flank the chosen insertion site, in a broad host range E. coli/P. aeruginosa vector. This plasmid may be transferred to a suitable P. aeruginosa (lacZM15+) strain, and the resulting strain infected by the previously described (lacZ+) Phi33 derivatives that have been modified to introduce the gene for SASP-C, codon optimised for expression in P. aeruginosa, under the control of a P. aeruginosa fda promoter. Progeny phage may be harvested and double recombinants identified by plaquing on P. aeruginosa (lacZM15+), looking for loss of the lacZ reporter on medium containing a chromogenic substrate that detects the action of -galactosidase.

Experimental Procedures

[0076] 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 (Tm) of the primers, according to manufacturers instructions. PCR reactions for screening purposes may be carried out using Taq DNA polymerase (NEB), depending upon the Tm of the primers, according to manufacturers 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).

[0077] 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.

[0078] All clonings, unless otherwise stated, may be achieved by ligating DNAs overnight with T4 DNA ligase and then transforming them into E. coli cloning strains, such as DH5 or TOP10, with isolation on selective medium, as described elsewhere (Sambrook et al., 1989).

[0079] An E. coli/P. aeruginosa broad host range vector, such as pSM1080A, may be used to transfer genes between E. coli and P. aeruginosa. pSM1080A was previously produced by combining the broad host-range origin of replication from a P. aeruginosa plasmid, oriT from pRK2, the tetAR selectable marker for use in both E. coli and P. aeruginosa, from plasmid pRK415, and the high-copy-number, E. coli origin of replication, oriV, from plasmid pUC19.

[0080] An E. coli vector that is unable to replicate in P. aeruginosa, pSM1104, may be used to generate P. aeruginosa mutants by allelic exchange. pSM1104 was previously produced by combining oriT from pRK2, the tetAR selectable marker for use in both E. coli and P. aeruginosa, from plasmid pRK415, the high-copy-number, E. coli origin of replication, oriV, from plasmid pUC19, and the sacB gene from Bacillus subtilis strain 168, under the control of a strong promoter, for use as a counter-selectable marker.

Detection of Phi33-Like Phage (PB1-Like Phage Family) Conserved N-Terminal Tail Fibre Regions by PCR

[0081] 1. Primers for the detection of Phi33-like phage-like tail fibre genes in experimental phage samples may be designed as follows:

[0082] 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.

[0083] 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.

TABLE-US-00001 B4500 (SEQIDNO:1) 5-GTGATCACACCCGAACTG-3 B4501 (SEQIDNO:2) 5-CGATGAAGAAGAGTTGGTTTTG-3 B4502 (SEQIDNO:3) 5-ACGCCGGACTACGAAATCAG-3 B4503 (SEQIDNO:4) 5-TCCGGAGACGTTGATGGT-3 B4504 (SEQIDNO:5) 5-CCTTTCATCGATTTCCACTTC-3 B4505 (SEQIDNO:6) 5-TTCGTGGACGCCCAGTCCCA-3

[0084] 2. Phi33-like tail fibre genes may be detected in experimental phage samples as follows:

[0085] 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.

Construction of Plasmids to Generate Pseudomonas aeruginosa Strains Carrying the Escherichia coli lacZM15 Gene, Immediately Downstream of the phoA Locus of the Bacterial Genome

[0086] 1. Plasmid pSMX200 (FIG. 1), comprising pSM1104 carrying DNA flanking the 3 end of the P. aeruginosa PAO1 phoA homologue, may be constructed as follows.

[0087] A region comprising the terminal approximately 1 kb of the phoA gene from P. aeruginosa may be amplified by PCR using primers B4200 and B4201 (FIG. 1). The PCR product may then be cleaned and digested with SpeI and BglII. A second region comprising approximately 1 kb downstream of the phoA gene from P. aeruginosa, including the 3 end of the PA3297 open reading frame, may be amplified by PCR using primers B4202 and B4203 (FIG. 1). This second PCR product may then be cleaned and digested with BglII and XhoI. The two digests may be cleaned again and ligated to pSM1104 that has been digested with SpeI and XhoI, in a 3-way ligation, to yield plasmid pSMX200 (FIG. 1).

[0088] Primer B4200 consists of a 5 SpeI restriction site (underlined), followed by sequence located approximately 1 kb upstream of the stop codon of phoA from P. aeruginosa strain PAO1 (FIG. 1). Primer B4201 consists of 5 BglII and AflII restriction sites (underlined), followed by sequence complementary to the end of the phoA gene from P. aeruginosa strain PA01 (the stop codon is in lower case; FIG. 1). Primer B4202 consists of 5 BglII and NheI restriction sites (underlined), followed by sequence immediately downstream of the stop codon of the phoA gene from P. aeruginosa strain PAO1 (FIG. 1). Primer B4203 consists of a 5 XhoI restriction site (underlined), followed by sequence within the PA3297 open reading frame, approximately 1 kb downstream of the phoA gene from P. aeruginosa strain PAO1 (FIG. 1).

TABLE-US-00002 PrimerB4200 (SEQIDNO:7) 5-gataACTAGTCCTGGTCCACCGGGGTCAAG-3 PrimerB4201 (SEQIDNO:8) 5-gctcagatcttccttaagtcaGTCGCGCAGGTTCAG-3 PrimerB4202 (SEQIDNO:9) 5-aggaagatctgagctagcTCGGACCAGAACGAAAAAG-3 PrimerB4203 (SEQIDNO:10) 5-gataCTCGAGGCGGATGAACATTGAGGTG-3

[0089] 2. Plasmid pSMX203 (FIG. 1), comprising pSMX200 carrying lacZM15 under the control of a lac promoter, may be constructed as follows.

[0090] The lacZM15 gene under the control of a lac promoter may be amplified by PCR from Escherichia coli strain DH10B using primers B4208 and B4209 (FIG. 1). The resulting PCR product may then be digested with BglII and NheI, and ligated to pSMX200 that has also been digested with BglII and NheI, to yield plasmid pSMX203 (FIG. 1).

[0091] Primer B4208 consists of a 5 BglII restriction site (underlined), followed by sequence of the lac promoter (FIG. 1). Primer B4209 consists of a 5 NheI restriction site (underlined), followed by a bi-directional transcriptional terminator and sequence complementary to the 3 end of lacZM15 (underlined, in bold; FIG. 1).

TABLE-US-00003 PrimerB4208 (SEQIDNO:11) 5-gataagatctgagcgcaacgcaattaatgtg-3 PrimerB4209 (SEQIDNO:12) 5-gatagctagcAGTCAAAAGCCTCCGGTCGGAGGCTTTTGACTTTATT TTTGACACCAGACCAAC-3

[0092] Genetic modification of Pseudomonas aeruginosa to introduce the Escherichia coli lacZM15 gene immediately downstream of the phoA locus of the bacterial genome [0093] 1. Plasmid pSMX203 (FIG. 1) may be transferred to P. aeruginosa by conjugation, selecting for primary recombinants by acquisition of resistance to tetracycline (50 g/ml). [0094] 2. Double recombinants may then be selected via sacB-mediated counterselection, by plating onto medium containing 10% sucrose. [0095] 3. Isolates growing on 10% sucrose may then be screened by PCR to confirm that lacZM15 has been introduced downstream of the P. aeruginosa phoA gene. [0096] 4. Following verification of an isolate (PAX21), this strain may then be used as a host for further modification of bacteriophage, where complementation of a lacZ reporter is required.
Construction of a Plasmid to Replace the 3 Section of the Phi33 Tail Fibre with that of PTP92, Utilising a lacZ Screening Process

[0097] 1. pSMX284 (FIG. 2), comprising pSM1080A carrying the region immediately downstream of the Phi33 tail fibre gene, may be constructed as follows.

[0098] A 1 kb region of Phi33 sequence covering the terminal 20 bases of the Phi33 tail fibre, and the adjacent downstream region, may be amplified by PCR using primers B4222 and B4249 (FIG. 2). The resulting PCR product may then be cleaned and digested with NheI, and ligated to pSM1080A that has also been digested with NheI and then treated with alkaline phosphatase prior to ligation, yielding plasmid pSMX284 (FIG. 2).

[0099] Primer B4222 consists of a 5 NheI restriction site (underlined), followed by sequence from Phi33, approximately 1 kb downstream of the end of the Phi33 tail fibre gene (FIG. 2). B4249 consists of 5 NheI-KpnI-AvrII restriction sites (underlined), followed by sequence complementary to the 3 end of the Phi33 tail fibre and sequence immediately downstream of the tail fibre open reading frame (FIG. 2).

TABLE-US-00004 B4222 (SEQIDNO:13) 5-gataGCTAGCATGGTTTTCACGACCATG-3 B4249 (SEQIDNO:14) 5-GATAGCTAGCGAGGTACCGACCTAGGTTTTCCAGCGAGTGACGTAA AATG-3

[0100] 2. pSMX285 (FIG. 2), comprising pSMX284 carrying lacZ, a 3 section of the PTP92 tail fibre gene sequence, and a region of Phi33 sequence comprising the 5 end of the tail fibre gene and sequence located immediately upstream of the Phi33 tail fibre gene, may be constructed as follows.

[0101] The lacZ open reading frame may be amplified by PCR from pUC19 using primers B4250 and B4252 (FIG. 2). The PTP92 tail fibre 3 section may be amplified by PCR from PTP92 using primers B4251 and B4254 (FIG. 2). The lacZ open reading frame may then be joined to the PTP92 tail fibre gene 3 section by SOEing PCR using the outer primers, B4250 and B4254. A region comprising sequence of the 5 end of the Phi33 tail fibre gene, and sequence located immediately upstream of the Phi33 tail fibre gene, may be amplified by PCR using primers B4253 and B4229 (FIG. 2). This PCR product may then be joined to the PCR product comprising lacZ and the PTP92 tail fibre gene 3 section, by SOEing PCR using the outer primers B4250 and B4229. The resulting PCR product may then be cleaned and digested with AvrII and KpnI, and ligated to pSMX284 that has also been digested with AvrII and KpnI, yielding plasmid pSMX285 (FIG. 2).

[0102] Primer B4250 consists of a 5 AvrII restriction site, followed by sequence complementary to the 3 end of the lacZ open reading frame (FIG. 2). Primer B4252 consists of a 5 section of sequence that overlaps the 3 end of the PTP92 tail fibre gene (underlined), followed by sequence of the 5 end of the lacZ open reading frame. Primer B4251 is the reverse complement of primer B4252 (FIG. 2). Primer B4254 consists of 5 sequence from within the Phi33 tail fibre gene (underlined), followed by sequence within the 3 end of the PTP92 tail fibre gene (FIG. 2). Primer B4253 is the reverse complement of Primer B4254. Primer B4229 consists of a 5 KpnI restriction site (underlined), followed by sequence that is complementary to a region approximately 1 kb upstream of the tail fibre gene in Phi33 (FIG. 2).

TABLE-US-00005 PrimerB4250 (SEQIDNO:15) 5-GataCCTAGGttagcgccattcgccattc-3 PrimerB4252 (SEQIDNO:16) 5-CTATTCCAGCGGGTAACGTAAAatgaccatgattacggattC-3 PrimerB4251 (SEQIDNO:17) 5-GaatccgtaatcatggtcatTTTACGTTACCCGCTGGAATAG-3 PrimerB4254 (SEQIDNO:18) 5-CAAGCGGGCCGGCTGGTCTCTCGGCAATAACTCCTATGTGATC-3 PrimerB4253 (SEQIDNO:19) 5-GATCACATAGGAGTTATTGCCGAGAGACCAGCCGGCCCGCTTG-3 PrimerB4229 (SEQIDNO:20) 5-gataGGTACCGCGACCGGTCTGTACTTC-3

[0103] Genetic modification of Phi33 to replace the 3 section of the tail fibre gene with that of PTP92 [0104] 1. Plasmid pSMX285 (FIG. 2; FIG. 3) may be introduced into P. aeruginosa strain PAX21 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 g/ml), yielding strain PTA80. [0105] 2. Strain PTA80 may be infected with phage Phi33, and the progeny phage harvested. [0106] 3. Recombinant phage in which the 3 end of the Phi33 tail fibre gene has been replaced by that of PTP92, and to which lacZ has been added, may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX21, onto medium containing S-gal, looking for black plaques, which are indicative of -galactosidase activity. [0107] 4. PCR may be carried out to check that the tail fibre gene has been replaced, and that lacZ is present. [0108] 5. Following identification of a verified isolate (PTPX81; FIG. 3), this isolate may be plaque purified twice more on P. aeruginosa strain PAX21, prior to further use.
Construction of a Plasmid to Replace the 3 Section of the Phi33 Tail Fibre with that of PTP47, Utilising a lacZ Screening Process

[0109] 1. pSMX286 (FIG. 2), comprising pSMX284 carrying lacZ, a 3 section of the PTP47 tail fibre gene sequence, and a region of Phi33 sequence comprising the 5 end of the tail fibre gene and sequence located immediately upstream of the Phi33 tail fibre gene, may be constructed as follows.

[0110] The lacZ open reading frame may be amplified by PCR from pUC19 using primers B4250 and B4258 (FIG. 2). The PTP47 tail fibre 3 section may be amplified by PCR from PTP47 using primers B4259 and B4260 (FIG. 2). The lacZ open reading frame may then be joined to the PTP47 tail fibre gene 3 section by SOEing PCR using the outer primers, B4250 and B4260. A region comprising sequence of the 5 end of the Phi33 tail fibre gene, and sequence located immediately upstream of the Phi33 tail fibre gene, may be amplified by PCR using primers B4261 and B4229 (FIG. 2). This PCR product may then be joined to the PCR product comprising lacZ and the PTP47 tail fibre gene 3 section, by SOEing PCR using the outer primers B4250 and B4229. The resulting PCR product may then be cleaned and digested with AvrII and KpnI, and ligated to pSMX284 that has also been digested with AvrII and KpnI, yielding plasmid pSMX286 (FIG. 2).

[0111] Primer B4250 consists of a 5 AvrII restriction site, followed by sequence complementary to the 3 end of the lacZ open reading frame (FIG. 2). Primer B4258 consists of a 5 section of sequence that overlaps the 3 end of the PTP47 tail fibre gene (underlined), followed by sequence of the 5 end of the lacZ open reading frame. Primer B4259 is the reverse complement of primer B4258 (FIG. 2). Primer B4260 consists of 5 sequence from within the Phi33 tail fibre gene (underlined), followed by sequence within the 3 end of the PTP47 tail fibre gene (FIG. 2). Primer B4261 is the reverse complement of Primer B4260. Primer B4229 consists of a 5 KpnI restriction site (underlined), followed by sequence that is complementary to a region approximately 1 kb upstream of the tail fibre gene in Phi33 (FIG. 2).

TABLE-US-00006 PrimerB4250 (SEQIDNO:15) 5-GataCCTAGGttagcgccattcgccattc-3 PrimerB4258 (SEQIDNO:21) 5-CTTTTCCAGCGAGTGACGTAAAatgaccatgattacggattC-3 PrimerB4259 (SEQIDNO:22) 5-gaatccgtaatcatggtcatTTTACGTCACTCGCTGGAAAAG-3 PrimerB4260 (SEQIDNO:23) 5-CAAGCGGGCCGGCTGGTCTCTCGGCAATAACTCCTATGTGATC-3 PrimerB4261 (SEQIDNO:24) 5-GATCACATAGGAGTTATTGCCGAGAGACCAGCCGGCCCGCTTG-3 PrimerB4229 (SEQIDNO:20) 5-gataGGTACCGCGACCGGTCTGTACTTC-3
Genetic Modification of Phi33 to Replace the 3 Section of the Tail Fibre Gene with that of PTP47 [0112] 1. Plasmid pSMX286 (FIG. 2; FIG. 4) may be introduced into P. aeruginosa strain PAX21 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 g/ml), yielding strain PTA81. [0113] 2. Strain PTA81 may be infected with phage Phi33, and the progeny phage harvested. [0114] 3. Recombinant phage in which the 3 end of the Phi33 tail fibre gene has been replaced by that of PTP47, and to which lacZ has been added, may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX21, onto medium containing S-gal, looking for black plaques, which are indicative of -galactosidase activity. [0115] 4. PCR may be carried out to check that the tail fibre gene has been replaced, and that lacZ is present. [0116] 5. Following identification of a verified isolate (PTPX82; FIG. 4), this isolate may be plaque purified twice more on P. aeruginosa strain PAX21, prior to further use.
Construction of a Plasmid to Remove the lacZ Marker from PTPX81

[0117] 1. pSMX287 (FIG. 5), comprising pSM1080A carrying a 3 section of the PTP92 tail fibre gene, and a region of Phi33 sequence located immediately downstream of the Phi33 tail fibre gene, may be constructed as follows.

[0118] The region of Phi33 sequence located immediately downstream of the Phi33 tail fibre may be amplified by PCR using primers B4222 and B4255 (FIG. 5). The 3 end of the PTP92 tail fibre gene may be amplified by PCR using primers B4256 and B4257 (FIG. 5). These two PCR products may then be joined by SOEing PCR, using the two outer primers B4222 and B4257. The resulting PCR product may then be cleaned, digested with NheI, cleaned again, and ligated to pSM1080A that has also been digested with NheI and then treated with alkaline phosphatase prior to ligation, to yield plasmid pSMX287 (FIG. 5).

[0119] Primer B4255 consists of a 5 section of the end of the PTP92 tail fibre gene (underlined), followed by sequence immediately downstream of the Phi33 tail fibre gene (FIG. 5). Primer B4256 is the reverse complement of primer B4255 (FIG. 5). Primer B4257 consists of a 5 NheI restriction site (underlined), followed by sequence of the terminal 1 kb of the PTP92 tail fibre gene (FIG. 5).

TABLE-US-00007 PrimerB4255 (SEQIDNO:25) 5-CTATTCCAGCGGGTAACGTAAAATGAAATGGACGCGGATCAG-3 PrimerB4256 (SEQIDNO:26) 5-CTGATCCGCGTCCATTTCATTTTACGTTACCCGCTGGAATAG-3 PrimersB4257 (SEQIDNO:27) 5-gataGCTAGCGGCAATAACTCCTATGTGATC-3
Genetic Modification of PTPX81 to Remove the lacZ Marker [0120] 1. Plasmid pSMX287 (FIG. 5; FIG. 3) may be introduced into P. aeruginosa strain PAX21 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 g/ml), yielding strain PTA82. [0121] 2. Strain PTA82 may be infected with phage PTPX81, and the progeny phage harvested. [0122] 3. Recombinant phage in which the lacZ marker has been removed may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX21, onto medium containing S-gal, looking for white plaques, which are indicative of loss of -galactosidase activity. [0123] 4. PCR may be carried out to check that the tail fibre gene has been retained, and that lacZ has been removed. [0124] 5. Following identification of a verified isolate (PTPX83; FIG. 3), this isolate may be plaque purified twice more on P. aeruginosa strain PAX21, prior to further use.
Construction of a Plasmid to Remove the lacZ Marker from PTPX82

[0125] 1. pSMX288 (FIG. 5), comprising pSM1080A carrying a 3 section of the PTP47 tail fibre gene, and a region of Phi33 sequence located immediately downstream of the Phi33 tail fibre gene, may be constructed as follows.

[0126] The region of Phi33 sequence located immediately downstream of the Phi33 tail fibre may be amplified by PCR using primers B4222 and B4262 (FIG. 5). The 3 end of the PTP47 tail fibre gene may be amplified by PCR using primers B4263 and B4264 (FIG. 5). These two PCR products may then be joined by SOEing PCR, using the two outer primers B4222 and B4264. The resulting PCR product may then be cleaned, digested with NheI, cleaned again, and ligated to pSM1080A that has also been digested with NheI and then treated with alkaline phosphatase prior to ligation, to yield plasmid pSMX288 (FIG. 5).

[0127] Primer B4262 consists of a 5 section of the end of the PTP47 tail fibre gene (underlined), followed by sequence immediately downstream of the Phi33 tail fibre gene (FIG. 5). Primer B4263 is the reverse complement of primer B4262 (FIG. 5). Primer B4264 consists of a 5 NheI restriction site (underlined), followed by sequence of the terminal 1 kb of the PTP47 tail fibre gene (FIG. 5).

TABLE-US-00008 PrimerB4262 (SEQIDNO:28) 5-CTTTTCCAGCGAGTGACGTAAAATGAAATGGACGCGGATCAG-3 PrimerB4263 (SEQIDNO:29) 5-CTGATCCGCGTCCATTTCATTTTACGTCACTCGCTGGAAAAG-3 PrimersB4264 (SEQIDNO:30) 5-gataGCTAGCGGCAATAACTCCTATGTGATC-3
Genetic Modification of PTPX82 to Remove the lacZ Marker [0128] 1. Plasmid pSMX288 (FIG. 5; FIG. 4) may be introduced into P. aeruginosa strain PAX21 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 g/ml), yielding strain PTA83. [0129] 2. Strain PTA83 may be infected with phage PTPX82, and the progeny phage harvested. [0130] 3. Recombinant phage in which the lacZ marker has been removed may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX21, onto medium containing S-gal, looking for white plaques, which are indicative of loss of -galactosidase activity. [0131] 4. PCR may be carried out to check that the tail fibre gene has been retained, and that lacZ has been removed. [0132] 5. Following identification of a verified isolate (PTPX84; FIG. 4), this isolate may be plaque purified twice more on P. aeruginosa strain PAX21, prior to further use.

[0133] Construction of a plasmid to introduce rpsB-SASP-C and lacZ into the phage genome of Phi33, PTPX83, PTPX84, and similar phage

[0134] 1. Plasmid pSMX251 (FIG. 6), comprising pSM1080A containing regions of Phi33 flanking the chosen insertion site for rpsB-SASP-C, such as the intergenic region immediately downstream of the tail fibre operon, may be constructed as follows.

[0135] The region of Phi33 sequence immediately downstream of the chosen insertion site may be amplified by PCR using primers B4900 and B4901 (FIG. 6). This PCR product may then be cleaned and digested with NheI and AvrII. The region of Phi33 sequence immediately upstream of the chosen insertion site may be amplified by PCR using primers B4902 and B4903 (FIG. 6). This second PCR product may then be cleaned and digested with AvrII and NheI. The two PCR product digests may then be cleaned again and ligated to pSM1080A that has been digested with NheI and treated with alkaline phosphatase prior to ligation. Clones carrying one insert of each of the two PCR products may be identified by PCR using primers B4900 and B4903, and NheI restriction digest analysis of the purified putative clones, to identify plasmid pSMX251 (FIG. 6).

[0136] Primer B4900 consists of a 5 NheI restriction site (underlined), followed by Phi33 sequence located approximately 500 bp downstream of the Phi33 insertion site that is within the intergenic region immediately downstream of the tail fibre operon (FIG. 6). Primer B4901 consists of 5 AvrII and XhoI restriction sites (underlined), followed by sequence of Phi33 that is complementary to sequence located immediately downstream of the Phi33 insertion site (FIG. 6). Primer B4902 consists of a 5 AvrII restriction site (underlined), followed by Phi33 sequence located immediately upstream of the insertion site (FIG. 6). Primer B4903 consists of a 5 NheI site (underlined), followed by Phi33 sequence that is complementary to sequence located approximately 500 bp upstream of the Phi33 insertion site (FIG. 6).

TABLE-US-00009 PrimerB4900 (SEQIDNO:31) 5gatagctagcTTTCTCGTTTTAATGTCG3 PrimerB4901 (SEQIDNO:32) 5gataCCTAGGtgCTCGAGTATTCGCCCAAAAGAAAAG3 PrimerB4902 (SEQIDNO:33) 5gataCCTAGGTCAGGAGCCTTGATTGATC3 PrimerB4903 (SEQIDNO:34) 5gatagctagcGGACTGGTAAGTCTGGTG3

[0137] 2. Plasmid pSMX252 (FIG. 6), comprising pSMX251 containing SASP-C under the control of an rpsB promoter, may be constructed as follows.

[0138] The SASP-C gene from Bacillus megaterium strain KM (ATCC 13632) may be amplified by PCR using primers B4904 and B4270 (FIG. 6). The resulting PCR product may then be digested with XhoI and NcoI. The rpsB promoter may be amplified by PCR from P. aeruginosa using primers B4271 and B4905 (FIG. 6). The resulting PCR product may then be digested with NcoI and AvrII. The two digested PCR products may then be cleaned and ligated to pSMX251 that has been digested with XhoI and AvrII, yielding plasmid pSMX252 (FIG. 6).

[0139] Primer B4904 comprises a 5 XhoI restriction site, followed by 5 bases, and then a bi-directional transcriptional terminator, and then sequence complementary to the 3 end of the SASP-C gene from B. megaterium strain KM (ATCC 13632) (underlined, in bold; FIG. 6). Primer B4270 comprises a 5 NcoI restriction site (underlined), followed by sequence of the 5 end of the SASP-C gene from B. megaterium strain KM (ATCC 13632) (FIG. 6). Primer B4271 comprises a 5 NcoI restriction site (underlined), followed by sequence complementary to the end of the rpsB promoter from P. aeruginosa PA01 (FIG. 6). Primer B4905 comprises a 5 AvrII restriction site (underlined), followed by sequence of the beginning of the rpsB promoter from P. aeruginosa PAO1 (FIG. 6).

TABLE-US-00010 PrimerB4904 (SEQIDNO:35) 5-gataCTCGAGGATCTAGTCAAAAGCCTCCGACCGGAGGCTTTTGACT ttagtacttgccgcctag-3 PrimerB4270 (SEQIDNO:36) 5-gataccATGGcaaattatcaaaacgcatc-3 PrimerB4271 (SEQIDNO:37) 5-gataCCATggTAGTTCCTCGATAAGTCG-3 PrimerB4905 (SEQIDNO:38) 5-gataCCTAGGCCTAGGgatctGACCGACCGATCTACTCC-3

[0140] 3. pSMX253 (FIG. 6), comprising pSMX252 containing lacZ, may be constructed as follows.

[0141] lacZ may be PCR amplified using primers B4906 and B4907 (FIG. 6). The resulting PCR product may then be digested with XhoI and ligated to pSMX252 that has also been digested with XhoI and treated with alkaline phosphatase prior to ligation, to yield pSMX253 (FIG. 6).

[0142] Primer B4906 consists of a 5 XhoI restriction site (underlined), followed by sequence complementary to the 3 end of lacZ (FIG. 6). Primer B4907 consists of a 5 XhoI restriction site (underlined), followed by sequence of the lac promoter driving expression of lacZ (FIG. 6).

TABLE-US-00011 PrimerB4906 (SEQIDNO:39) 5-gataCTCGAGttagcgccattcgccattc-3 PrimerB4907 (SEQIDNO:40) 5-gataCTCGAGgcgcaacgcaattaatgtg-3
Genetic Modification of Phi33, PTPX83, PTPX84, and Similar Phage, to Introduce rpsB-SASP-C and lacZ [0143] 1. Plasmid pSMX253 (FIG. 6; FIG. 3; FIG. 4; FIG. 7) may be introduced into P. aeruginosa strain PAX21 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 g/ml), yielding strain PTA51. [0144] 2. Strain PTA51 may be infected in individual experiments with phage Phi33, or PTPX83, or PTPX84, or other similar phage, and the progeny phage harvested. [0145] 3. Recombinant phage, in which rpsB-SASP-C and lacZ have been introduced into the chosen insertion site, may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX21, onto medium containing S-gal, looking for black plaques, which are indicative of -galactosidase activity. [0146] 4. PCR may be carried out to check that rpsB-SASP-C and lacZ are present. [0147] 5. Following identification of verified isolates (for example, PTPX85 (FIG. 7), PTPX86 (FIG. 3), PTPX87 (FIG. 4)), the isolates may be plaque purified twice more on P. aeruginosa strain PAX21, prior to further use.
Genetic Modification to Remove the lacZ Marker from PTPX85, PTPX86, PTPX87, and Similar Derivatives of Phi33 [0148] 1. Plasmid pSMX252 (FIG. 6; FIG. 3; FIG. 4; FIG. 7) may be introduced into P. aeruginosa strain PAX21 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 g/ml), yielding strain PTA85. [0149] 2. Strain PTA85 may be infected in individual experiments with phage PTPX85, or PTPX86, or PTPX87, or other similar phage, and the progeny phage harvested. [0150] 3. Recombinant phage, in which lacZ marker has been removed, may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX21, onto medium containing S-gal, looking for white plaques, which are indicative of loss of -galactosidase activity. [0151] 4. PCR may be carried out to confirm removal of the lacZ marker, while ensuring that rpsB-SASP-C is still present. [0152] 5. Following identification of verified isolates (for example, PTPX88 (FIG. 7), PTPX89 (FIG. 3), PTPX90 (FIG. 4)), the isolates may be plaque purified twice more on P. aeruginosa strain PAX21, prior to further use.

[0153] Construction of a plasmid to introduce fda-SASP-C (codon optimised) and lacZ into a chosen insertion site, located in an intergenic region immediately downstream of the tail fibre operon, within the genome of Phi33, PTPX83, PTPX84, and similar phage

[0154] 1. Plasmid pSMX254 (FIG. 8), comprising pSMX251 containing SASP-C codon optimised for expression in P. aeruginosa, under the control of an fda promoter, may be constructed as follows.

[0155] The SASP-C gene from Bacillus megaterium strain KM (ATCC 13632) may be codon optimised for expression in P. aeruginosa (FIG. 9) and synthesised in vitro. The codon optimised SASP-C gene may then be amplified by PCR using primers B4312 and B4313 (FIG. 8). The fda promoter may be amplified by PCR from P. aeruginosa using primers B4314 and B4315 (FIG. 8). The resulting two PCR products may then be joined by splicing by overlap extension (SOEing) PCR, using the outer primers B4312 and B4314 (FIG. 8). The resulting fda-codon optimised SASP-C-terminator PCR product may then be digested with XhoI and AvrII, cleaned, and ligated to pSMX251 that has been digested with XhoI and AvrII, yielding plasmid pSMX254 (FIG. 8).

[0156] Primer B4312 comprises a 5 XhoI restriction site, followed by a bi-directional transcriptional terminator, and then sequence complementary to the 3 end of the SASP-C gene from B. megaterium strain KM (ATCC 13632) that has been codon optimised for expression in P. aeruginosa (underlined, in bold; FIG. 8). Primer B4313 comprises sequence of the 3 end of the fda promoter from P. aeruginosa PA01 (in bold) followed by sequence of the 5 end of the codon optimised SASP-C gene. Primer B4314 comprises sequence complementary to the 5 end of the codon optimised SASP-C gene followed by sequence complementary to the 3 end of the fda promoter from P. aeruginosa PAO1 (FIG. 8). Primer B4315 comprises a 5 AvrII restriction site (underlined), followed by sequence of the beginning of the fda promoter from P. aeruginosa PAO1 (FIG. 8).

TABLE-US-00012 PrimerB4312 (SEQIDNO:41) 5-gataCTCGAGAGTCAAAAGCCTCCGACCGGAGGCTTTTGACTTCAGT ACTTGCCGCCCAG-3 PrimerB4313 (SEQIDNO:42) 5-GATTGGGAGATACGAGAACCATGGCCAACTACCAGAACGC-3 PrimerB4314 (SEQIDNO:43) 5-GCGTTCTGGTAGTTGGCCATGGTTCTCGTATCTCCCAATC-3 PrimerB4315 (SEQIDNO:44) 5-GATACCTAGGAACGACGAAGGCCTGGTG-3

[0157] 3. pSMX255 (FIG. 8), comprising pSMX254 containing lacZ, may be constructed as follows.

[0158] lacZ may be PCR amplified using primers B4906 and B4907 (FIG. 8). The resulting PCR product may then be digested with XhoI and ligated to pSMX254 that has also been digested with XhoI and treated with alkaline phosphatase prior to ligation, to yield pSMX255 (FIG. 8).

[0159] Primer B4906 consists of a 5 XhoI restriction site (underlined), followed by sequence complementary to the 3 end of lacZ (FIG. 8). Primer B4907 consists of a 5 XhoI restriction site (underlined), followed by sequence of the lac promoter driving expression of lacZ (FIG. 8).

TABLE-US-00013 PrimerB4906 (SEQIDNO:40) 5-gataCTCGAGttagcgccattcgccattc-3 PrimerB4907 (SEQIDNO:41) 5-gataCTCGAGgcgcaacgcaattaatgtg-3
Genetic Modification of Phi33, PTPX83, PTPX84, and Similar Phage, to Introduce Fda-Codon Optimised SASP-C and lacZ [0160] 1. Plasmid pSMX255 (FIG. 8; FIG. 10; FIG. 11; FIG. 12) may be introduced into P. aeruginosa strain PAX21 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 g/ml), yielding strain PTA86. [0161] 2. Strain PTA86 may be infected in individual experiments with phage Phi33, or PTPX83, or PTPX84, or other similar phage, and the progeny phage harvested. [0162] 3. Recombinant phage, into which fda-codon optimised SASP-C and lacZ have been introduced, may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX21, onto medium containing S-gal, looking for black plaques, which are indicative of -galactosidase activity. [0163] 4. PCR may be carried out to check that fda-codon optimised SASP-C and lacZ are present. [0164] 5. Following identification of verified isolates (for example, PTPX91 (FIG. 12), PTPX92 (FIG. 10), PTPX93 (FIG. 11)), the isolates may be plaque purified twice more on P. aeruginosa strain PAX21, prior to further use.
Genetic Modification to Remove the lacZ Marker from PTPX91, PTPX92, PTPX93, and Similar Derivatives of Phi33 [0165] 1. Plasmid pSMX254 (FIG. 8; FIG. 10; FIG. 11; FIG. 12) may be introduced into P. aeruginosa strain PAX21 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 g/ml), yielding strain PTA87. [0166] 2. Strain PTA87 may be infected in individual experiments with phage PTPX91, or PTPX92, or PTPX93, or other similar phage, and the progeny phage harvested. [0167] 3. Recombinant phage, in which lacZ marker has been removed, may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX21, onto medium containing S-gal, looking for white plaques, which are indicative of loss of -galactosidase activity. [0168] 4. PCR may be carried out to confirm removal of the lacZ marker, while ensuring that fda-codon optimised SASP-C is still present. [0169] 5. Following identification of verified isolates (for example, PTP387 (FIG. 12), PTP388 (FIG. 10), PTP389 (FIG. 11)), the isolates may be plaque purified twice more on P. aeruginosa strain PAX21, prior to further use.

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