Patent Application: US-201515517664-A

Abstract:
modified bacteriophage , uses thereof , and compositions containing the modified bacteriophage are described . the compositions are useful for human treatment and may treat various conditions , including bacterial infections .

Description:
this invention will now be described in more detail , by way of example only , and with reference to the accompanying drawings , in which : fig1 is a schematic diagram showing construction of plasmids containing laczδm15 and phi33 endolysin ; fig2 is a schematic diagram showing construction of plasmids with replaced tail fibre sections ; fig3 is a schematic diagram showing construction of phage with hybrid tail fibre genes , which may be subsequently modified to have endolysin replaced by sasp - c according to the invention ; fig4 is a schematic diagram showing construction of phage with further hybrid tail fibre genes , which may be subsequently modified to have endolysin replaced by sasp - c according to the invention ; fig5 is a schematic diagram showing construction of bacteriophage with hybrid tail fibre genes , in which the laczα marker has been removed ; fig6 is a schematic diagram showing construction of plasmids in which the endolysin gene is replaced by sasp - c ; fig7 is a schematic diagram showing production of further bacteriophage according to the invention ; fig8 is a schematic diagram showing construction of plasmids in which the endolysin gene is replaced by sasp - c codon optimised for expression in p . aeruginosa ; fig9 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 ; fig1 is a schematic diagram showing production of bacteriophage in which the endolysin gene is replaced by sasp - c which has been codon optimised for expression in p . aeruginosa ; fig1 is a schematic diagram showing production of further bacteriophage according to the invention ; fig1 is a schematic diagram showing production of further bacteriophage according to the invention ; fig1 is a clustal 2 . 1 multiple sequence alignment of the tail fibre genes from phage spm - 1 , f8 , pb1 , c36 , lbl3 , phi33 , lma2 , kpp12 , jg024 , ptp92 , nh - 4 , 14 - 1 , ptp47 , sn ; and fig1 shows a bar graph of kill of p . aeruginosa by phi33 , ptp92 and a mixture of ptp110 and ptp114 . fig1 . 24 hour time - kill curve showing the in vitro efficacy of pt3 . 8 against p . aeruginosa strains 3503 ( a ) and atcc 27853 ( b ). cultures were grown in luria bertani ( lb ) broth supplemented with 5 mm calcium chloride , 5 mm magnesium sulphate and 0 . 1 % glucose , at 37 ° c . fig1 . 60 minute time - kill curve showing the in vitro efficacy of pt3 . 8 against p . aeruginosa strain 3503 . cultures were grown in luria bertani ( lb ) broth supplemented with 5 mm calcium chloride , 5 mm magnesium sulphate and 0 . 1 % glucose , at 37 ° c . fig1 . in vivo efficacy of pt3 . 8 in a murine lung model of infection . line labelled “ 0 hour ” shows viable cell numbers in the lung tissue at the time of treatment with either vehicle ( tris buffered saline containing 1 mm magnesium sulphate , 10 mm calcium chloride and 10 % v / v glycerol ) or pt3 . 8 ( 2 hours post infection with p . aeruginosa ). the viable cell counts in lung tissue at 2 and 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 . for the 22 hour vehicle group , accurate values could be ascertained for only 3 of 6 animals , the viable cell counts for the other 3 animals were & gt ; 10 ̂ 11 cfu / ml . generic product covering a single tail fibre within an individual phage , or a mix of phages where each type of phage has a single , different tail fibre summary of the genetic modification of a lytic bacteriophage to render it non - lytic , and 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 30s ribosomal subunit protein s2 gene ( rpsb ). 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 . 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 the resulting phage may be rendered non - lytic , and 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 . 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 laczα 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 laczα , may be isolated by plaquing on a suitable p . aeruginosa ( laczδm15 + ) host strain using medium containing a chromogenic substrate that detects the action of β - galactosidase . in a subsequent step , the laczα 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 laczα 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 laczα marker . recombinants that retain the new tail fibre or tail fibre section , but from which laczα has been removed , may be isolated by plaquing on a suitable p . aeruginosa ( laczδm15 + ) host strain using medium containing a chromogenic substrate that detects the action of β - galactosidase . in a subsequent step , a similar homologous recombination may be used to replace the endolysin gene of phi33 , or of any of the previously described phi33 derivatives , or similar bacteriophage or similar derivatives , with the gene for sasp - c , under the control of a p . aeruginosa rpsb promoter , while simultaneously adding an e . coli laczα reporter gene for the identification of recombinant phage . a region consisting of sasp - c controlled by the rpsb promoter , and the e . coli laczα allele , may be cloned between two regions of phi33 that flank the endolysin gene , in a broad host range e . coli / p . aeruginosa vector . this plasmid may be transferred to a suitable p . aeruginosa ( endolysin + laczδm15 + ) strain , and the resulting strain infected by phi33 or the previously constructed phi33 derivative ( from which the initial laczα marker has been removed ). progeny phage may be harvested and double recombinants identified by plaquing on p . aeruginosa ( endolysin + laczδm15 + ), looking for acquisition of the new laczα reporter on medium containing a chromogenic substrate that detects the action of β - galactosidase . in an alternative subsequent step , a similar homologous recombination may be used to replace the endolysin gene of phi33 , or of any of the previously described phi33 derivatives , or similar bacteriophage or similar derivatives , with 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 laczα reporter gene for the identification of recombinant phage . a region consisting of codon optimised sasp - c controlled by the fda promoter , and the e . coli laczα allele , may be cloned between two regions of phi33 that flank the endolysin gene , in a broad host range e . coli / p . aeruginosa vector . this plasmid may be transferred to a suitable p . aeruginosa ( endolysin + laczδm15 + ) strain , and the resulting strain infected by phi33 or the previously constructed phi33 derivative ( from which the initial laczα marker has been removed ). progeny phage may be harvested and double recombinants identified by plaquing on p . aeruginosa ( endolysin + laczδm15 + ), looking for acquisition of the new laczα reporter on medium containing a chromogenic substrate that detects the action of β - galactosidase . 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 either the e . coli laczδm15 gene at a suitable location in the bacterial genome , or both the phi33 endolysin gene and the e . coli laczδm15 at a suitable location in the bacterial genome , to complement the laczα or δendolysin , laczαphenotypes 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 endolysin and laczδm15 transgenes 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 . the e . coli laczδm15 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 μg / ml ) resistance . isolates ( laczδm15 + ) 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 ). the phi33 endolysin gene and the e . coli laczδm15 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 μg / ml ) resistance . isolates ( endolysin + , laczδm15 + ) 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 ). in a subsequent step , a similar homologous recombination may be used to remove the laczα marker from the previously described , ( laczα + ) phi33 derivatives that have been modified to replace the endolysin gene with 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 endolysin gene , in a broad host range e . coli / p . aeruginosa vector . this plasmid may be transferred to a suitable p . aeruginosa ( endolysin + laczδm15 k ) strain , and the resulting strain infected by the previously described ( laczα + ) phi33 derivatives that have been modified to replace the endolysin gene with 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 ( endolysin + laczδm15 + ), looking for loss of the laczα reporter on medium containing a chromogenic substrate that detects the action of β - galactosidase . in an alternative subsequent step , a similar homologous recombination may be used to remove the laczα marker from the previously described , ( laczα + ) phi33 derivatives that have been modified to replace the endolysin gene with 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 endolysin gene , in a broad host range e . coli / p . aeruginosa vector . this plasmid may be transferred to a suitable p . aeruginosa ( endolysin + laczδm15 + ) strain , and the resulting strain infected by the previously described ( laczα + ) phi33 derivatives that have been modified to replace the endolysin gene with 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 ( endolysin + laczδm15 + ), looking for loss of the laczα reporter on medium containing a chromogenic substrate that detects the action of β - galactosidase . 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 m ) of the primers , according to manufacturers instructions . pcr reactions for screening purposes may be carried out using taq dna polymerase ( neb ), depending upon the t m 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 β - galactosidase , s - gal , that upon digestion by β - galactosidase forms a black precipitate when chelated with ferric iron , may be purchased from sigma ( s9811 ). 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 . 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 dh5α or top10 , with isolation on selective medium , as described elsewhere ( sambrook et al ., 1989 ). an e . coli / p . aeruginosa broad host range vector , such as psm1080 , may be used to transfer genes between e . coli and p . aeruginosa . psm1080 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 . 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 1 . primers for the detection of phi33 - like phage - like tail fibre genes in experimental phage samples may be designed as follows : 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 &# 39 ; 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 194 bp - long region ; pair b4502 and b4503 , defining a 774 bp - long region ; and pair b4504 and b4505 , defining a 365 bp - long region . 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 . b4500 ( seq id no : 1 ) 5 ′- gtgatcacacccgaactg - 3 ′ b4501 ( seq id no : 2 ) 5 ′- cgatgaagaagagttggttttg - 3 ′ b4502 ( seq id no : 3 ) 5 ′- acgccggactacgaaatcag - 3 ′ b4503 ( seq id no : 4 ) 5 ′- tccggagacgttgatggt - 3 ′ b4504 ( seq id no : 5 ) 5 ′- cctttcatcgatttccacttc - 3 ′ b4505 ( seq id no : 6 ) 5 ′- ttcgtggacgcccagtccca - 3 ′ 2 . phi33 - like tail fibre genes may be detected in experimental phage samples as follows : 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 either the escherichia coli laczδm15 gene , or both the phi33 endolysin gene and the escherichia coli laczδm15 gene , immediately downstream of the phoa locus of the bacterial genome 1 . plasmid psmx200 ( fig1 ), comprising psm1104 carrying dna flanking the 3 ′ end of the p . aeruginosa pao1 phoa homologue , may be constructed as follows . 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 ( fig1 ). 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 ( fig1 ). 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 ( fig1 ). 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 ( fig1 ). 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 pao1 ( the stop codon is in lower case ; fig1 ). 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 ( fig1 ). 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 ( fig1 ). 2 . plasmid psmx201 ( fig1 ), comprising psmx200 carrying the phi33 endolysin gene under the control of an endolysin promoter , may be constructed as follows . the endolysin promoter may be amplified by pcr from phi33 using primers b4204 and b4205 ( fig1 ). the endolysin gene itself may be amplified by pcr from phi33 using primers b4206 and b4207 ( fig1 ). the two pcr products may then be joined together by splicing by overlap extension ( soeing ) pcr , using the two outer primers , b4204 and b4207 . the resulting pcr product may then be digested with aflii and bglii , and ligated to psmx200 that has also been digested with aflii and bglii , to yield plasmid psmx201 ( fig1 ). primer b4204 consists of a 5 ′ aflii restriction site ( underlined ), followed by a bi - directional transcriptional terminator ( soxr terminator , 60 - 96 bases of genbank accession number dq058714 ), and sequence of the beginning of the phi33 endolysin promoter region ( underlined , in bold ) ( fig1 ). primer b4205 consists of a 5 ′ region of sequence that is complementary to the region overlapping the start codon of the endolysin gene from phi33 , followed by sequence that is complementary to the end of the endolysin promoter region ( underlined , in bold ; fig1 ). primer b4206 is the reverse complement of primer b4205 ( see also fig1 ). primer b4207 consists of a 5 ′ bglii restriction site ( underlined ), followed by sequence complementary to the end of the phi33 endolysin gene ( fig1 ). primer b4204 ( seq id no : 11 ) 5 ′- gata cttaag aaaacaaactaaagcgcccttgtggcgctttagtttt atac tactgagaaaaatctggattc - 3 ′ primer b4205 ( seq id no : 12 ) 5 ′- gattttcatcaatactcctggatcc cgttaattcgaagagtcg - 3 ′ primer b4206 ( seq id no : 13 ) 5 ′- cgactcttcgaattaacg ggatccaggagtattgatgaaaatc - 3 ′ primer b4207 ( seq id no : 14 ) 5 ′- gata agatct tcaggagccttgattgatc - 3 ′ 3 . plasmid psmx202 ( fig1 ), comprising psmx201 carrying laczδm15 under the control of a lac promoter , may be constructed as follows . the laczδm15 gene under the control of a lac promoter may be amplified by pcr from escherichia coli strain dh10b using primers b4208 and b4209 ( fig1 ). the resulting pcr product may then be digested with bglii and nhei , and ligated to psmx201 that has also been digested with bglii and nhei , to yield plasmid psmx202 ( fig1 ). primer b4208 consists of a 5 ′ bglii restriction site ( underlined ), followed by sequence of the lac promoter ( fig1 ). 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 laczδm15 ( underlined , in bold ; fig1 ). primer b4208 ( seq id no : 15 ) 5 ′- gata agatct gagcgcaacgcaattaatgtg - 3 ′ primer b4209 ( seq id no : 16 ) 5 ′- gata gctagc agtcaaaagcctccggtcggaggcttttgact ttatt tttgacaccagaccaac - 3 ′ 4 . plasmid psmx203 ( fig1 ), comprising psmx200 carrying laczδm15 under the control of a lac promoter , may be constructed as follows . the laczδm15 gene under the control of a lac promoter may be amplified by pcr from escherichia coli strain dh10b using primers b4208 and b4209 ( fig1 ). 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 ( fig1 ). primer b4208 consists of a 5 ′ bglii restriction site ( underlined ), followed by sequence of the lac promoter ( fig1 ). 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 laczδm15 ( underlined , in bold ; fig1 ). primer b4208 ( seq id no : 15 ) 5 ′- gata agatct gagcgcaacgcaattaatgtg - 3 ′ primer b4209 ( seq id no : 16 ) 5 ′- gata gctagc agtcaaaagcctccggtcggaggcttttgact ttatt tttgacaccagaccaac - 3 ′ genetic modification of pseudomonas aeruginosa to introduce the phi33 endolysin gene and escherichia coli laczδm15 immediately downstream of the phoa locus of the bacterial genome 1 . plasmid psmx202 ( fig1 ) may be transferred to p . aeruginosa by conjugation , selecting for primary recombinants by acquisition of resistance to tetracycline ( 50 μg / ml ). 2 . double recombinants may then be selected via sacb - mediated counterselection , by plating onto medium containing 10 % sucrose . 3 . isolates growing on 10 % sucrose may then be screened by pcr to confirm that the endolysin gene and laczδm15 have been introduced downstream of the p . aeruginosa phoa gene . 4 . following verification of an isolate ( pax20 ), this strain may then be used as a host for further modification of phi33 - related bacteriophage , where complementation of both an endolysin mutation and a laczα reporter are required . genetic modification of pseudomonas aeruginosa to introduce the escherichia coli laczδm15 gene immediately downstream of the phoa locus of the bacterial genome 1 . plasmid psmx203 ( fig1 ) may be transferred to p . aeruginosa by conjugation , selecting for primary recombinants by acquisition of resistance to tetracycline ( 50 μg / ml ). 2 . double recombinants may then be selected via sacb - mediated counterselection , by plating onto medium containing 10 % sucrose . 3 . isolates growing on 10 % sucrose may then be screened by pcr to confirm that laczδm15 has been introduced downstream of the p . aeruginosa phoa gene . 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 laczα reporter is required . construction of a plasmid to replace the 3 ′ section of the phi33 tail fibre with that of ptp92 , utilising a laczα screening process 1 . psmx204 ( fig2 ), comprising psm1080 carrying the region immediately downstream of the phi33 tail fibre gene , may be constructed as follows . 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 ( fig2 ). the resulting pcr product may then be cleaned and digested with nhei , and ligated to psm1080 that has also been digested with nhei and then treated with alkaline phosphatase prior to ligation , yielding plasmid psmx204 ( fig2 ). 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 ( fig2 ). 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 ( fig2 ). b4222 ( seq id no : 17 ) 5 ′- gata gctagc atggttttcacgaccatg - 3 ′ b4249 ( seq id no : 18 ) 5 ′- gata gctagc ga ggtacc ga cctagg ttttccagcgagtgacgtaa aatg - 3 ′ 2 . psmx205 ( fig2 ), comprising psmx204 carrying laczα , 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 . the laczα open reading frame may be amplified by pcr from puc19 using primers b4250 and b4252 ( fig2 ). the ptp92 tail fibre 3 ′ section may be amplified by pcr from ptp92 using primers b4251 and b4254 ( fig2 ). the laczα 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 ( fig2 ). this pcr product may then be joined to the pcr product comprising laczα 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 psmx204 that has also been digested with avrii and kpni , yielding plasmid psmx205 ( fig2 ). primer b4250 consists of a 5 ′ avrii restriction site , followed by sequence complementary to the 3 ′ end of the laczα open reading frame ( fig2 ). 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 laczα open reading frame . primer b4251 is the reverse complement of primer b4252 ( fig2 ). 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 ( fig2 ). 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 ( fig2 ). primer b4250 ( seq id no : 19 ) 5 ′- gata cctagg ttagcgccattcgccattc - 3 ′ primer b4252 ( seq id no : 20 ) 5 ′- ctattccagcgggtaacgtaaa atgaccatgattacggattc - 3 ′ primer b4251 ( seq id no : 21 ) 5 ′- gaatccgtaatcatggtcat tttacgttacccgctggaatag - 3 ′ primer b4254 ( seq id no : 22 ) 5 ′- caagcgggccggctggtctctc ggcaataactcctatgtgatc - 3 ′ primer b4253 ( seq id no : 23 ) 5 ′- gatcacataggagttattgcc gagagaccagccggcccgcttg - 3 ′ primer b4229 ( seq id no : 24 ) 5 ′- gata ggtacc gcgaccggtctgtacttc - 3 ′ genetic modification of phi33 to replace the 3 ′ section of the tail fibre gene with that of ptp92 1 . plasmid psmx205 ( fig2 ; fig3 ) may be introduced into p . aeruginosa strain pax21 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta20 . 2 . strain pta20 may be infected with phage phi33 , and the progeny phage harvested . 3 . recombinant phage in which the 3 ′ end of the phi33 tail fibre gene has been replaced by that of ptp92 , and to which laczα 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 β - galactosidase activity . 4 . pcr may be carried out to check that the tail fibre gene has been replaced , and that laczα is present . 5 . following identification of a verified isolate ( ptpx21 ; fig3 ), 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 laczα screening process 1 . psmx206 ( fig2 ), comprising psmx204 carrying laczα , 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 . the laczα open reading frame may be amplified by pcr from puc19 using primers b4250 and b4258 ( fig2 ). the ptp47 tail fibre 3 ′ section may be amplified by pcr from ptp47 using primers b4259 and b4260 ( fig2 ). the laczα 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 ( fig2 ). this pcr product may then be joined to the pcr product comprising laczα 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 psmx204 that has also been digested with avrii and kpni , yielding plasmid psmx206 ( fig2 ). primer b4250 consists of a 5 ′ avrii restriction site , followed by sequence complementary to the 3 ′ end of the laczα open reading frame ( fig2 ). 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 laczα open reading frame . primer b4259 is the reverse complement of primer b4258 ( fig2 ). 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 ( fig2 ). 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 ( fig2 ). primer b4250 ( seq id no : 19 ) 5 ′- gata cctagg ttagcgccattcgccattc - 3 ′ primer b4252 ( seq id no : 20 ) 5 ′- ctattccagcgggtaacgtaaa atgaccatgattacggattc - 3 ′ primer b4251 ( seq id no : 21 ) 5 ′- gaatccgtaatcatggtcat tttacgttacccgctggaatag - 3 ′ primer b4254 ( seq id no : 22 ) 5 ′- caagcgggccggctggtctctc ggcaataactcctatgtgatc - 3 ′ primer b4253 ( seq id no : 23 ) 5 ′- gatcacataggagttattgcc gagagaccagccggcccgcttg - 3 ′ primer b4229 ( seq id no : 24 ) 5 ′- gata ggtacc gcgaccggtctgtacttc - 3 ′ genetic modification of phi33 to replace the 3 ′ section of the tail fibre gene with that of ptp47 1 . plasmid psmx206 ( fig2 ; fig4 ) may be introduced into p . aeruginosa strain pax21 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta21 . 2 . strain pta21 may be infected with phage phi33 , and the progeny phage harvested . 3 . recombinant phage in which the 3 ′ end of the phi33 tail fibre gene has been replaced by that of ptp47 , and to which laczα 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 β - galactosidase activity . 4 . pcr may be carried out to check that the tail fibre gene has been replaced , and that laczα is present . 5 . following identification of a verified isolate ( ptpx22 ; fig4 ), this isolate may be plaque purified twice more on p . aeruginosa strain pax21 , prior to further use . construction of a plasmid to remove the laczα marker from ptpx21 1 . psmx207 ( fig5 ), comprising psm1080 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 . the region of phi33 sequence located immediately downstream of the phi33 tail fibre may be amplified by pcr using primers b4222 and b4255 ( fig5 ). the 3 ′ end of the ptp92 tail fibre gene may be amplified by pcr using primers b4256 and b4257 ( fig5 ). 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 psm1080 that has also been digested with nhei and then treated with alkaline phosphatase prior to ligation , to yield plasmid psmx207 ( fig5 ). 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 ( fig5 ). primer b4256 is the reverse complement of primer b4255 ( fig5 ). primer b4257 consists of a 5 ′ nhei restriction site ( underlined ), followed by sequence of the terminal 1 kb of the ptp92 tail fibre gene ( fig5 ). primer b4255 ( seq id no : 29 ) 5 ′- ctattccagcgggtaacgtaa aatgaaatggacgcggatcag - 3 ′ primer b4256 ( seq id no : 30 ) 5 ′- ctgatccgcgtccatttcatt ttacgttacccgctggaatag - 3 ′ primers b4257 ( seq id no : 31 ) 5 ′- gata gctagc ggcaataactcctatgtgatc - 3 ′ 1 . plasmid psmx207 ( fig5 ; fig3 ) may be introduced into p . aeruginosa strain pax21 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta22 . 2 . strain pta22 may be infected with phage ptpx21 , and the progeny phage harvested . 3 . recombinant phage in which the laczα 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 β - galactosidase activity . 4 . pcr may be carried out to check that the tail fibre gene has been retained , and that laczα has been removed . 5 . following identification of a verified isolate ( ptpx23 ; fig3 ), this isolate may be plaque purified twice more on p . aeruginosa strain pax21 , prior to further use . construction of a plasmid to remove the laczα marker from ptpx22 1 . psmx208 ( fig5 ), comprising psm1080 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 . the region of phi33 sequence located immediately downstream of the phi33 tail fibre may be amplified by pcr using primers b4222 and b4262 ( fig5 ). the 3 ′ end of the ptp47 tail fibre gene may be amplified by pcr using primers b4263 and b4264 ( fig5 ). 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 psm1080 that has also been digested with nhei and then treated with alkaline phosphatase prior to ligation , to yield plasmid psmx208 ( fig5 ). 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 ( fig5 ). primer b4263 is the reverse complement of primer b4262 ( fig5 ). primer b4264 consists of a 5 ′ nhei restriction site ( underlined ), followed by sequence of the terminal 1 kb of the ptp47 tail fibre gene ( fig5 ). primer b4262 ( seq id no : 32 ) 5 ′- cttttccagcgagtgacgtaa aatgaaatggacgcggatcag - 3 ′ primer b4263 ( seq id no : 33 ) 5 ′- ctgatccgcgtccatttcatt ttacgtcactcgctggaaaag - 3 ′ primers b4264 ( seq id no : 34 ) 5 ′- gata gctagc ggcaataactcctatgtgatc - 3 ′ 1 . plasmid psmx208 ( fig5 ; fig4 ) may be introduced into p . aeruginosa strain pax21 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta23 . 2 . strain pta23 may be infected with phage ptpx22 , and the progeny phage harvested . 3 . recombinant phage in which the laczα 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 β - galactosidase activity . 4 . pcr may be carried out to check that the tail fibre gene has been retained , and that laczα has been removed . 5 . following identification of a verified isolate ( ptpx24 ; fig4 ), this isolate may be plaque purified twice more on p . aeruginosa strain pax21 , prior to further use . construction of a plasmid to replace the endolysin gene of phi33 , ptpx23 , ptpx24 , and similar phage , by rpsb - sasp - c and laczα 1 . plasmid psmx209 ( fig6 ), comprising psm1080 containing regions of phi33 flanking the endolysin gene , may be constructed as follows . the region of phi33 sequence immediately downstream of the endolysin gene may be amplified by pcr using primers b4265 and b4266 ( fig6 ). this pcr product may then be cleaned and digested with ndei and nhei . the region of phi33 sequence immediately upstream of the endolysin gene may be amplified by pcr using primers b4267 and b4268 ( fig6 ). this second pcr product may then be cleaned and digested with ndei and nhei . the two pcr product digests may then be cleaned again and ligated to psm1080 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 b4265 and b4268 , and ndei restriction digest analysis of the purified putative clones , to identify plasmid psmx209 ( fig6 ). primer b4265 consists of a 5 ′ nhei restriction site ( underlined ), followed by phi33 sequence located approximately 340 bp downstream of the phi33 endolysin gene ( fig6 ). primer b4266 consists of 5 ′ ndei and kpni restriction sites ( underlined ), followed by sequence of phi33 that is located immediately downstream of the endolysin gene ( fig6 ). primer b4267 consists of a 5 ′ ndei restriction site ( underlined ), followed by sequence that is complementary to sequence located immediately upstream of the phi33 endolysin gene ( fig6 ). primer b4268 consists of a 5 ′ nhei site ( underlined ), followed by phi33 sequence that is located approximately 340 bp upstream of the endolysin gene ( fig6 ). primer b4265 ( seq id no : 35 ) 5 ′- gata gctagc ttggccagaaagaaggcg - 3 ′ primer b4266 ( seq id no : 36 ) 5 ′- gata catatg tc ggtacc tattcgcccaaaagaaaag - 3 ′ primer b4267 ( seq id no : 37 ) 5 ′- gata catatg tcaatactcctgatttttg - 3 ′ primer b4268 ( seq id no : 38 ) 5 ′- gata gctagc aatgaaatggacgcggatc - 3 ′ 2 . plasmid psmx210 ( fig6 ), comprising psmx209 containing sasp - c under the control of an rpsb promoter , may be constructed as follows . the sasp - c gene from bacillus megaterium strain km ( atcc 13632 ) may be amplified by pcr using primers b4269 and b4270 ( fig6 ). the resulting pcr product may then be digested with kpni and ncoi . the rpsb promoter may be amplified by pcr from p . aeruginosa using primers b4271 and b4272 ( fig6 ). the resulting pcr product may then be digested with ncoi and ndei . the two digested pcr products may then be cleaned and ligated to psmx209 that has been digested with kpni and ndei , yielding plasmid psmx210 ( fig6 ). primer b4269 comprises a 5 ′ kpni 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 ; fig6 ). 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 ) ( fig6 ). primer b4271 comprises a 5 ′ ncoi restriction site ( underlined ), followed by sequence complementary to the end of the rpsb promoter from p . aeruginosa pao1 ( fig6 ). primer b4272 comprises a 5 ′ ndei restriction site ( underlined ), followed by sequence of the beginning of the rpsb promoter from p . aeruginosa pao1 ( fig6 ). primer b4269 ( seq id no : 39 ) 5 ′- gata ggtacc gatctagtcaaaagcctccgaccggaggcttttgact ttagtacttgccgcctag - 3 ′ primer b4270 ( seq id no : 40 ) 5 ′- gata ccatgg caaattatcaaaacgcatc - 3 ′ primer b4271 ( seq id no : 41 ) 5 ′- gata ccatgg tagttcctcgataagtcg - 3 ′ primer b4272 ( seq id no : 42 ) 5 ′- gata catatg cctagggatctgaccgaccgatctactcc - 3 ′ 3 . psmx211 ( fig6 ), comprising psmx210 containing laczα , may be constructed as follows . laczα may be pcr amplified using primers b4273 and b4274 ( fig6 ). the resulting pcr product may then be digested with kpni and ligated to psmx210 that has also been digested with kpni and treated with alkaline phosphatase prior to ligation , to yield psmx211 ( fig6 ). primer b4273 consists of a 5 ′ kpni restriction site ( underlined ), followed by sequence complementary to the 3 ′ end of laczα ( fig6 ). primer b4274 consists of a 5 ′ kpni restriction site ( underlined ), followed by sequence of the lac promoter driving expression of laczα ( fig6 ). genetic modification of phi33 , ptpx23 , ptpx24 , and similar phage , to replace the endolysin gene with rpsb - sasp - c and laczα 1 . plasmid psmx211 ( fig6 ; fig3 ; fig4 ; fig7 ) may be introduced into p . aeruginosa strain pax20 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta24 . 2 . strain pta24 may be infected in individual experiments with phage phi33 , or ptpx23 , or ptpx24 , or other similar phage , and the progeny phage harvested . 3 . recombinant phage , in which the endolysin gene has been replaced by rpsb - sasp - c and laczα , may be identified by plaquing the lysate from step ( 2 ) on p . aeruginosa strain pax20 , onto medium containing s - gal , looking for black plaques , which are indicative of β - galactosidase activity . 4 . pcr may be carried out to check that the endolysin gene has been replaced , and that rpsb - sasp - c and laczα are present . 5 . following identification of verified isolates ( for example , ptpx25 ( fig7 ), ptpx26 ( fig3 ), ptpx27 ( fig4 )), the isolates may be plaque purified twice more on p . aeruginosa strain pax20 , prior to further use . genetic modification to remove the laczα marker from ptpx25 , ptpx26 , ptpx27 , and similar derivatives of phi33 1 . plasmid psmx210 ( fig6 ; fig3 ; fig4 ; fig7 ) may be introduced into p . aeruginosa strain pax20 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta25 . 2 . strain pta25 may be infected in individual experiments with phage ptpx25 , or ptpx26 , or ptpx27 , or other similar phage , and the progeny phage harvested . 3 . recombinant phage , in which laczα marker has been removed , may be identified by plaquing the lysate from step ( 2 ) on p . aeruginosa strain pax20 , onto medium containing s - gal , looking for white plaques , which are indicative of loss of β - galactosidase activity . 4 . pcr may be carried out to confirm removal of the laczα marker , while ensuring that rpsb - sasp - c is still present . 5 . following identification of verified isolates ( for example , ptp114 ( fig7 ), ptp110 ( fig3 ), ptpx28 ( fig4 )), the isolates may be plaque purified twice more on p . aeruginosa strain pax20 , prior to further use . construction of a plasmid to replace the endolysin gene of phi33 , ptpx23 , ptpx24 , and similar phage , by fda - sasp - c ( codon optimised ) and laczα 1 . plasmid psmx212 ( fig8 ), comprising psm1080 containing regions of phi33 flanking the endolysin gene , may be constructed as follows . the region of phi33 sequence immediately downstream of the endolysin gene may be amplified by pcr using primers b4265 and b4310 ( fig8 ). this pcr product may then be cleaned and digested with nhei and xhoi . the region of phi33 sequence immediately upstream of the endolysin gene may be amplified by pcr using primers b4311 and b4268 ( fig8 ). this second pcr product may then be cleaned and digested with xhoi and nhei . the two pcr product digests may then be cleaned again and ligated to psm1080 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 b4265 and b4268 , and xhoi restriction digest analysis of the purified putative clones , to identify plasmid psmx212 ( fig8 ). primer b4265 consists of a 5 ′ nhei restriction site ( underlined ), followed by phi33 sequence located approximately 340 bp downstream of the phi33 endolysin gene ( fig8 ). primer b4310 consists of 5 ′ avrii and xhoi restriction sites ( underlined ), followed by sequence of phi33 that is located immediately downstream of the endolysin gene ( fig8 ). primer b4311 consists of 5 ′ xhoi and avrii restriction sites ( underlined ), followed by sequence that is complementary to sequence located immediately upstream of the phi33 endolysin gene ( fig8 ). primer b4268 consists of a 5 ′ nhei site ( underlined ), followed by phi33 sequence that is located approximately 340 bp upstream of the endolysin gene ( fig8 ). primer b4265 ( seq id no : 35 ) 5 ′- gatagctagcttggccagaaagaaggcg - 3 ′ primer b4310 ( seq id no : 45 ) 5 ′- gatacctaggtcctcgagtattcgcccaaaagaaaag - 3 ′ primer b4311 ( seq id no : 46 ) 5 ′- gatactcgaggacctaggtcaatactcctgatttttg - 3 ′ primer b4268 ( seq id no : 38 ) 5 ′- gatagctagcaatgaaatggacgcggatc - 3 ′ 2 . plasmid psmx213 ( fig8 ), comprising psmx212 containing sasp - c codon optimised for expression in p . aeruginosa , under the control of an fda promoter , may be constructed as follows . the sasp - c gene from bacillus megaterium strain km ( atcc 13632 ) may be codon optimised for expression in p . aeruginosa ( fig9 ) and synthesised in vitro . the codon optimised sasp - c gene may then be amplified by pcr using primers b4312 and b4313 ( fig8 ). the fda promoter may be amplified by pcr from p . aeruginosa using primers b4314 and b4315 ( fig8 ). the resulting two pcr products may then be joined by splicing by overlap extension ( soeing ) pcr , using the outer primers b4312 and b4314 ( fig8 ). the resulting fda - codon optimised sasp - c - terminator pcr product may then be digested with xhoi and avrii , cleaned , and ligated to psmx212 that has been digested with xhoi and avrii , yielding plasmid psmx213 ( fig8 ). 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 ; fig8 ). primer b4313 comprises sequence of the 3 ′ end of the fda promoter from p . aeruginosa pao1 ( 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 ( fig8 ). primer b4315 comprises a 5 ′ avrii restriction site ( underlined ), followed by sequence of the beginning of the fda promoter from p . aeruginosa pao1 ( fig8 ). primer b4312 ( seq id no : 47 ) 5 ′- gata ctcgag agtcaaaagcctccgaccggaggcttttgact tcagt acttgccgcccag - 3 ′ primer b4313 ( seq id no : 48 ) 5 ′- gattgggagatacgagaacc atggccaactaccagaacgc - 3 ′ primer b4314 ( seq id no : 49 ) 5 ′- gcgttctggtagttggccat ggttctcgtatctcccaatc - 3 ′ primer b4315 ( seq id no : 50 ) 5 ′- gata cctagg aacgacgaaggcctggtg - 3 ′ 3 . psmx214 ( fig8 ), comprising psmx213 containing laczα , may be constructed as follows . laczα may be pcr amplified using primers b4316 and b4317 ( fig8 ). the resulting pcr product may then be digested with xhoi and ligated to psmx213 that has also been digested with xhoi and treated with alkaline phosphatase prior to ligation , to yield psmx214 ( fig8 ). primer b4316 consists of a 5 ′ xhoi restriction site ( underlined ), followed by sequence complementary to the 3 ′ end of laczα ( fig8 ). primer b4317 consists of a 5 ′ xhoi restriction site ( underlined ), followed by sequence of the lac promoter driving expression of laczα ( fig8 ). genetic modification of phi33 , ptpx23 , ptpx24 , and similar phage , to replace the endolysin gene with fda - codon optimised sasp - c and laczα 1 . plasmid psmx214 ( fig8 ; fig1 ; fig1 ; fig1 ) may be introduced into p . aeruginosa strain pax20 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta26 . 2 . strain pta26 may be infected in individual experiments with phage phi33 , or ptpx23 , or ptpx24 , or other similar phage , and the progeny phage harvested . 3 . recombinant phage , in which the endolysin gene has been replaced by fda - codon optimised sasp - c and laczα , may be identified by plaquing the lysate from step ( 2 ) on p . aeruginosa strain pax20 , onto medium containing s - gal , looking for black plaques , which are indicative of β - galactosidase activity . 4 . pcr may be carried out to check that the endolysin gene has been replaced , and that fda - codon optimised sasp - c and laczα are present . 5 . following identification of verified isolates ( for example , ptpx29 ( fig1 ), ptpx30 ( fig1 ), ptpx34 ( fig1 )), the isolates may be plaque purified twice more on p . aeruginosa strain pax20 , prior to further use . genetic modification to remove the laczα marker from ptpx29 , ptpx30 , ptpx34 , and similar derivatives of phi33 1 . plasmid psmx213 ( fig8 ; fig1 ; fig1 ; fig1 ) may be introduced into p . aeruginosa strain pax20 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta27 . 2 . strain pta27 may be infected in individual experiments with phage ptpx29 , or ptpx30 , or ptpx34 , or other similar phage , and the progeny phage harvested . 3 . recombinant phage , in which laczα marker has been removed , may be identified by plaquing the lysate from step ( 2 ) on p . aeruginosa strain pax20 , onto medium containing s - gal , looking for white plaques , which are indicative of loss of β - galactosidase activity . 4 . pcr may be carried out to confirm removal of the laczα marker , while ensuring that fda - codon optimised sasp - c is still present . 5 . following identification of verified isolates ( for example , ptp284 ( fig1 ), ptp384 ( fig1 ), ptp385 ( fig1 )), the isolates may be plaque purified twice more on p . aeruginosa strain pax20 , prior to further use . abedon s t . 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