Patent Application: US-201515517732-A

Abstract:
a method for modifying the genome of a lytic target phage , uses of the method and products thereof are described . compositions comprising such phage are also described . the compositions may be formulated as a medicament , which are useful for human treatment and may treat various conditions , including bacterial infections .

Description:
this invention will now be described in further 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 an endolysin gene ; fig2 is a schematic diagram showing construction of plasmids with replaced tail fibre sections , for the genetic modification of phage to add sasp - c , or sasp - c in addition to a lacza marker ; fig3 is a schematic diagram showing production of phage in which sasp - c , or sasp - c in addition to a lacza marker , have been added to the phage , by recombination , using hords as a means of selecting for recombinant phage ; fig4 is a schematic diagram showing construction of plasmids with replaced tail fibre sections , for the genetic modification of phage to replace the endolysin gene by sasp - c , or by sasp - c and lacza marker ; fig5 is a schematic diagram showing production of phage in which the endolysin gene has been deleted and replaced by sasp - c , or by sasp - c and a lacza marker , by recombination , using hords as a means of selecting for recombinant phage ; and fig6 shows multiple sequence alignment of the tail fibre genes of related phages . the following examples are given to show the utility of the hords technique in adding exogenous dna to an obligately lytic phage . as an example , a dna region comprising the tail fibre gene , or section of a tail fibre gene , from an alternative phage , and the sasp - c gene from bacillus megaterium controlled by a pseudomonas aeruginosa fda promoter , may be cloned between two regions of phi33 dna that flank the native tail fibre region , or section thereof , 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of progeny phage , double recombinant bacteriophage in which the native phi33 tail fibre , or tail fibre section , has been replaced by the new tail fibre or tail fibre section , and to which the fda - sasp - c region of dna has been introduced , may be isolated by plaquing on a suitable p . aeruginosa strain that is a host for the new , recombinant bacteriophage , but is not a host for phi33 . as another example , for the construction of a phi33 derivative in which two , unrelated sections of foreign dna has been introduced into the genome , it is shown here as an example only , how the existing tail fibre , or section thereof , may be replaced by an alternative tail fibre or tail fibre section from a different bacteriophage , while simultaneously adding a sasp - c gene from bacillus megaterium under the control of a pseudomonas aeruginosa fda promoter , alongside a lacza marker from escherichia coli , via homologous recombination . a dna region comprising the tail fibre gene , or section of a tail fibre gene , from an alternative phage , the sasp - c gene from bacillus megaterium controlled by a pseudomonas aeruginosa fda promoter , and an escherichia coli lacza reporter gene , may be cloned between two regions of phi33 dna that flank the native tail fibre region , or section thereof , 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 recombinant bacteriophage in which the native phi33 tail fibre , or tail fibre section , has been replaced by the new tail fibre or tail fibre section , and to which the fda - sasp - c and lacza regions have been introduced , may be isolated by plaquing on a suitable p . aeruginosa strain that is a host for the new , recombinant bacteriophage , but is not a host for phi33 . if visualisation of the lacza marker is required , the pseudomonas aeruginosa host strain used should carry the escherichia coli laczδm15 allele at a suitable location in the host strain genome . as another example , for construction of a phi33 derivative in which a region of the bacteriophage genome is deleted , while simultaneously introducing a section of foreign dna into the genome , it is shown here as an example only , how the existing tail fibre , or section thereof , may be replaced by an alternative tail fibre or tail fibre section from a different bacteriophage , while simultaneously deleting the native endolysin gene to render the phage non - lytic , and also simultaneously introducing a sasp - c gene from bacillus megaterium under the control of a pseudomonas aeruginosa fda promoter , via homologous recombination . successful recombinants may be identified by selection of bacteriophage that plaque on a p . aeruginosa strain that is a host for the recombinant phage that carry the new host range determinant , but which is not a host for the original native phage , and which has also been modified such that the phi33 endolysin gene is present on the p . aeruginosa genome . a dna region comprising the tail fibre gene , or section of a tail fibre gene , from an alternative phage , and the sasp - c gene from bacillus megaterium controlled by a pseudomonas aeruginosa fda promoter , may be cloned between two regions of phi33 dna that flank the native tail fibre region and endolysin region , or section thereof , 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 recombinant bacteriophage in which the native phi33 tail fibre , or tail fibre section , has been replaced by the new tail fibre or tail fibre section , and to which the fda - sasp - c region of dna has been introduced , and from which the native endolysin gene has been deleted , may be isolated by plaquing on a suitable p . aeruginosa ( endolysin + ) strain that is a host for the new , recombinant bacteriophage , but is not a host for phi33 . in order to generate a non - lytic version of a lytic bacteriophage by this method , a suitable pseudomonas aeruginosa host strain is required that is a host for the recombinant bacteriophage that carries the new host range determinant , but that is not a host for the native bacteriophage , but in addition , carries the bacteriophage endolysin gene at a suitable location in the pseudomonas aeruginosa genome . similarly , if visualisation of a bacteriophage - bourne lacza reporter is required , a pseudomonas aeruginosa host strain is required that is a host for the recombinant bacteriophage that carries the new host range determinant , but that is not a host for the native bacteriophage , but in addition , carries the escherichia coli laczδm15 allele at a suitable location . the genomic location for insertion of transgenes such as these should be chosen such that no essential genes are affected and no unwanted phenotypes are generated as a result of polar effects on the expression of adjacent genes . as an example , one such a location could include immediately downstream of the phoa gene of pseudomonas aeruginosa . as an example , the phi33 endolysin gene 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 / m1 ) 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 ). as an example , the escherichia 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 ). 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 f3 - 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 a broad host - range origin of replication to allow replication in p . aeruginosa , 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 . construction of plasmids to generate pseudomonas aeruginosa strains carrying either the phi33 endolysin gene , or the escherichia coli laczδm15 gene , immediately downstream of the phoa locus of the bacterial genome 1 . plasmid psmx301 ( 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 b4300 and b4301 ( fig1 ). the pcr product may then be cleaned and digested with spel and bg1ii . a second region comprising approximately 1 kb downstream of the phoa gene from p . aeruginosa may be amplified by pcr using primers b4302 and b4303 ( fig1 ). this second pcr product may then be cleaned and digested with bg1ii and xhol . the two digests may be cleaned again and ligated to psm1104 that has been digested with spel and xhol , in a 3 - way ligation , to yield plasmid psmx301 ( fig1 ). primer b4300 consists of a 5 ′ spel restriction site ( underlined ), followed by sequence located approximately 1 kb upstream of the stop codon of phoa from p . aeruginosa strain pao1 ( fig1 ). primer b4301 consists of 5 ′ bg1ll and af1ll 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 b4302 consists of 5 ′ bg1ll 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 b4303 consists of a 5 ′ xhol restriction site ( underlined ), followed by sequence that is complementary to sequence approximately 1 kb downstream of the phoa gene from p . aeruginosa strain pao1 ( fig1 ). 2 . plasmid psmx302 ( fig1 ), comprising psmx301 carrying the endolysin gene from phi33 , under the control of an endolysin gene promoter , may be constructed as follows . the endolysin gene promoter may be amplified by pcr from phi33 using primers b4304 and b4305 ( fig1 ). the endolysin gene itself may be amplified by pcr from phi33 using primers b4306 and b4307 ( fig1 ). the two pcr products may then be joined together by splicing by overlap extension ( soeing ) pcr , using the two outer primers , b4304 and b4307 . the resulting pcr product may then be digested with af1ll and bg1ll , and ligated to psmx301 that has also been digested with af1ll and bg1ll , to yield plasmid psmx302 ( fig1 ). primer b4304 consists of a 5 ′ af1ll 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 endolysin promoter region ( underlined , in bold ) ( fig1 ). primer b4305 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 b4306 is the reverse complement of primer b4305 ( see also fig1 ). primer b4307 consists of a 5 ′ bg1ll restriction site ( underlined ), followed by sequence complementary to the end of the phi33 endolysin gene ( fig1 ). 3 . plasmid psmx303 , comprising psmx301 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 b4308 and b4309 ( fig1 ). the resulting pcr product may then be digested with bg1ll and nhei , and ligated to psmx301 that has also been digested with bg1ll and nhei , to yield plasmid psmx303 ( fig1 ). primer b4308 consists of a 5 ′ bg1ll restriction site ( underlined ), followed by sequence of the lac promoter ( fig1 ). primer b4309 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 ). genetic modification of pseudomonas aeruginosa to introduce the phi33 endolysin gene immediately downstream of the phoa locus of the bacterial genome 1 . plasmid psmx302 ( fig1 ) may be transferred to a suitable p . aeruginosa strain that is a host for bacteriophage carrying the new host range determinant , but which is not a host for the original bacteriophage , by conjugation , selecting for primary recombinants by acquisition of resistance to tetracycline ( 50 μg / m1 ). 2 . double recombinants may then be selected via sacb - mediated counter - selection , by plating onto medium containing 10 % sucrose . 3 . isolates growing on 10 % sucrose may then be screened by pcr to confirm that the phi33 endolysin gene has been introduced downstream of the p . aeruginosa phoa gene . 4 . following verification of an isolate ( pax31 ), this strain may then be used as a host for further modification of bacteriophage , where complementation of an endolysin mutation is required . genetic modification of pseudomonas aeruginosa to introduce the escherichia coli laczδm15 allele immediately downstream of the phoa locus of the bacterial genome 1 . plasmid psmx303 ( fig1 ) may be transferred to a suitable p . aeruginosa strain that is a host for bacteriophage carrying the new host range determinant , but which is not a host for the original bacteriophage , by conjugation , selecting for primary recombinants by acquisition of resistance to tetracycline ( 50 μg / m1 ). 2 . double recombinants may then be selected via sacb - mediated counter - selection , by plating onto medium containing 10 % sucrose . 3 . isolates growing on 10 % sucrose may then be screened by pcr to confirm that the escherichia coli laczδm15 allele has been introduced downstream of the p . aeruginosa phoa gene . 4 . following verification of an isolate ( pax32 ), this strain may then be used as a bacteriophage host , when complementation of a lacza reporter is desired . construction of a plasmid to introduce a new section of dna ( fda - sasp - c ) into the genome of bacteriophage phi33 , utilising an alternative host range determinant as a selectable marker . 1 . plasmid psmx304 ( fig2 ), comprising psm1080 carrying phi33 sequences flanking sequences of the tail fibre host range determinant of the related bacteriophage ptp92 , may be constructed as follows . the region immediately downstream of the phi33 tail fibre may be amplified by pcr using primers b4333 and b4334 ( fig2 ). the region encoding the c - terminal , receptor - binding region of the tail fibre of bacteriophage ptp92 may be amplified by pcr using primers b4335 and b4336 ( fig2 ). these two pcr products may then be joined by soeing pcr using the outer primers b4333 and b4336 . the region encoding the phi33 tail fibre n - terminal region , and the region immediately upstream of the phi33 tail fibre , may be amplified by pcr using primers b4337 and b4338 ( fig2 ). this phi33 tail fibre region may then be joined to the pcr product comprising the region downstream of the phi33 tail fibre and the ptp92 host range determinant , by soeing pcr using the outer primers b4333 and b4338 . the resulting pcr product may then be cleaned , digested with nhei , cleaned again and then ligated to psm1080 that has been digested with nhei , treated with alkaline phosphatase and cleaned , prior to ligation . this construction yields plasmid psmx304 ( fig2 ). primer b4333 consists of 5 ′ nhei - af1ii - paci restriction sites ( underlined ) followed by sequence complementary to a region approximately 1 kb downstream of the phi33 tail fibre ( fig2 ). primer b4334 consists of 5 ′ sequence of the end of the region encoding the c - terminal , receptor binding region of the ptp92 tail fibre , followed by sequence immediately downstream of the phi33 tail fibre ( underlined ; fig2 ). primer b4335 is the reverse complement of primer b4334 . primer b4336 consists of 5 ′ sequence complementary to a region that encodes part of the c - terminal , receptor binding region of the ptp92 tail fibre , followed by sequence complementary to the phi33 tail fibre ( underlined ; fig2 ). primer b4337 is the reverse complement of primer b4336 ( fig2 ). primer b4338 consists of a 5 ′ nhei restriction site ( underlined ), followed by sequence of a region upstream of the phi33 tail fibre ( fig2 ). 2 . plasmid psmx305 ( fig2 ), comprising psmx304 carrying a region of phi33 dna immediately downstream of the endolysin gene , the location chosen here for insertion of the fda - sasp - c foreign dna , may be constructed as follows . an approximately 1 kb region of phi33 dna located immediately downstream of the endolysin gene , the location chosen for insertion of the fda - sasp - c foreign dna , may be amplified by pcr using primers b4339 and b4340 ( fig2 ). the resulting pcr product may then be digested with af1ii and paci , cleaned , and ligated to psmx304 that has also been digested with af1ii and paci and cleaned , prior to ligation , yielding plasmid psmx305 ( fig2 ). primer b4339 consists of a 5 ′ af1ll restriction site ( underlined ), followed by phi33 sequence approximately 1 kb downstream of the location chosen here for insertion of the fda - sasp - c dna ( fig2 ). primer b4340 consists of 5 ′ paci - kpni - saci restriction sites ( underlined ), followed by sequence complementary to phi33 sequence located immediately downstream of the location chosen for insertion of the fda - sasp - c dna ( fig2 ). 3 . plasmid psmx306 ( fig2 ), comprising psmx305 carrying fda - sasp - c , may be constructed as follows . the sasp - c gene from bacillus megaterium strain km ( atcc 13632 ) may be amplified by pcr using primers b4341 and b4342 ( fig2 ). the resulting pcr product may then be cleaned , digested with kpni and ncoi , and cleaned again . the pseudomonas aeruginosa fda promoter may be amplified by pcr using primers b4343 and b4344 ( fig2 ). the resulting pcr product may then be cleaned , digested with ncoi and paci , and cleaned again . the two pcr products may then be ligated , in a 3 - way ligation , to psmx305 that has been digested with kpni and paci and cleaned prior to ligation , to yield plasmid psmx306 ( fig2 ). primer b4341 consists of a 5 ′ kpni restriction site ( underlined ), followed by a bi - directional transcription terminator ( tonb terminator ), followed by sequence complementary to the end of sasp - c from bacillus megaterium strain km ( atcc 13632 ) ( underlined , in bold ; fig2 ). primer b4342 ( fig2 ) consists of a 5 ′ ncoi restriction site ( underlined ), followed by sequence of the beginning of the sasp - c gene from bacillus megaterium strain km ( atcc 13632 ). primer b4343 consists of a 5 ′ ncoi restriction site ( underlined ), followed by sequence of the fda promoter ( fig2 ). primer b4344 consists of a 5 ′ paci restriction site ( underlined ), followed by sequence complementary to the fda promoter ( fig2 ). genetic modification of phi33 to add fda - sasp - c to the bacteriophage genome , utilising the ptp92 host range determinant as a means of selection 1 . plasmid psmx306 ( fig2 ) may be introduced into a p . aeruginosa strain that is a host for both the original , and the host range determinant donor phage , by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / m1 ), yielding strain pta31 . 2 . strain pta31 may be infected with phage phi33 , and the progeny phage harvested . 3 . recombinant phage , in which the ptp92 host range determinant has been transferred to phi33 , may be identified by plaquing the lysate from step ( 2 ) on p . aeruginosa strain 2726 , which is a host for the recombinant phage that carries the ptp92 host range determinant , but which is not a host for the parental bacteriophage phi33 . 4 . a pcr screen may be further carried out to identify isolates that have simultaneously acquired fda - sasp - c , in addition to the host range determinant from ptp92 . 5 . following identification of a verified isolate ( ptpx31 ; fig2 ; fig3 ), this isolate may be plaque purified twice more , prior to further use . construction of a plasmid to introduce two new sections of dna ( fda - sasp - c and lacza ) into the genome of bacteriophage phi33 , utilising an alternative host range determinant as a selectable marker . 1 . plasmid psmx307 ( fig2 ), comprising psmx306 carrying the lacza reporter from plasmid puc19 , may be constructed as follows . the lacza reporter may be amplified by pcr from puc19 using primers b4345 and b4346 ( fig2 ). the resulting pcr product may then be cleaned , digested with saci and kpni , cleaned again , and ligated to psmx306 that has been digested with saci and kpni and cleaned prior to ligation , to yield plasmid psmx307 ( fig2 ). primer b4345 consists of a 5 ′ saci restriction site ( underlined ), followed by sequence complementary to the 3 ′ end of lacza ( fig2 ). primer b4346 consists of a 5 ′ kpni restriction site ( underlined ), followed by sequence of the lac promoter ( fig2 ). genetic modification of phi33 to add fda - sasp - c and lacza to the bacteriophage genome , utilising the ptp92 host range determinant as a means of selection 1 . plasmid psmx307 ( fig2 ; fig3 ) may be introduced into a p . aeruginosa strain that is a host for both the original , and the host range determinant donor phage , by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / m1 ), yielding strain pta32 . 2 . strain pta32 may be infected with phage phi33 , and the progeny phage harvested . 3 . recombinant phage , in which the ptp92 host range determinant has been transferred to phi33 , may be identified by plaquing the lysate from step ( 2 ) on p . aeruginosa strain 2726 , which is a host for the recombinant phage that carries the ptp92 host range determinant , but which is not a host for the parental bacteriophage phi33 . 4 . a pcr screen may be further carried out to identify isolates that have simultaneously acquired fda - sasp - c and lacza , in addition to the host range determinant from ptp92 . 5 . isolates that have acquired lacza may further be confirmed by plaquing on pseudomonas aeruginosa strain pax32 ( fig1 ), using culture medium containing the chromogenic substrate for f3 - galactosidase , s - gal ( sigma ). bacteriophage which carry the lacza reporter generate black plaques on this host , using this culture medium , while plaques of bacteriophage lacking the lacza reporter remain clear . 6 . following identification of a verified isolate ( ptpx32 ; fig3 ), this isolate may be plaque purified twice more on , prior to further use . construction of a plasmid to simultaneously delete a section of bacteriophage phi33 ( the endolysin gene ) and introduce a new section of dna ( fda - sasp - c ) into the genome of bacteriophage phi33 , utilising an alternative host range determinant as a selectable marker . 1 . plasmid psmx308 ( fig4 ), comprising psm1080 carrying phi33 sequences flanking sequences of the tail fibre host range determinant of the related bacteriophage ptp92 , but including a deletion of the endolysin gene , may be constructed as follows . the region immediately downstream of the phi33 tail fibre may be amplified by pcr using primers b4347 and b4334 ( fig4 ). the region encoding the c - terminal , receptor - binding region of bacteriophage ptp92 may be amplified by pcr using primers b4335 and b4336 ( fig4 ). these two pcr products may then be joined by soeing pcr using the outer primers b4347 and b4336 . the phi33 sequence that encodes the n - terminal region of the phi33 tail fibre , and the tail fibre upstream sequence may be amplified from phi33 by pcr using primers b4337 and b4338 . this phi33 tail fibre region may then be joined to the pcr product comprising the region downstream of the phi33 tail fibre and the ptp92 host range determinant , by soeing pcr using the outer primers b4347 and b4338 . the resulting pcr product may then be cleaned , digested with nhei , cleaned again and then ligated to psm1080 that has been digested with nhei , treated with alkaline phosphatase and cleaned , prior to ligation . this construction yields plasmid psmx308 ( fig4 ). primer b4347 consists of 5 ′ nhei - af1ii - paci restriction sites ( underlined ) followed by sequence complementary to a region approximately 1 kb downstream of the phi33 tail fibre ( fig4 ). primer b4334 consists of 5 ′ sequence of the ptp92 host range determinant , followed by sequence immediately downstream of the phi33 tail fibre ( underlined ; fig4 ). primer b4335 is the reverse complement of primer b4334 ( fig4 ). primer b4336 consists of 5 ′ sequence complementary to the ptp92 host range determinant , followed by sequence complementary to the phi33 tail fibre ( underlined ; fig4 ). primer b4337 is the reverse complement of primer b4336 ( fig4 ). primer b4338 consists of a 5 ′ nhei restriction site ( underlined ), followed by sequence of a region upstream of the phi33 tail fibre ( fig4 ). 2 . plasmid psmx309 ( fig4 ), comprising psmx308 carrying a region of phi33 dna immediately downstream of the endolysin gene , which is the location chosen here for insertion of the fda - sasp - c foreign dna , may be constructed as follows . an approximately 1 kb region of phi33 dna located immediately downstream of the endolysin gene , the location chosen here for insertion of the fda - sasp - c foreign dna , may be amplified by pcr using primers b4339 and b4340 ( fig4 ). the resulting pcr product may then be digested with af1ll and paci , cleaned , and ligated to psmx308 that has also been digested with af1ll and paci and cleaned , prior to ligation , yielding plasmid psmx309 ( fig4 ). primer b4339 consists of a 5 ′ af1ll restriction site ( underlined ), followed by phi33 sequence approximately 1 kb downstream of the endolysin gene , the location chosen here for insertion of the fda - sasp - c dna ( fig4 ). primer b4340 consists of 5 ′ paci - kpni - saci restriction sites ( underlined ), followed by sequence complementary to phi33 sequence located immediately downstream of the endolysin gene , the location chosen here for insertion of the fda - sasp - c dna ( fig4 ). 3 . plasmid psmx310 ( fig4 ), comprising psmx309 carrying fda - sasp - c , may be constructed as follows . the sasp - c gene from bacillus megaterium strain km ( atcc 13632 ) may be amplified by pcr using primers b4341 and b4342 ( fig4 ). the resulting pcr product may then be cleaned , digested with kpni and ncoi , and cleaned again . the pseudomonas aeruginosa fda promoter may be amplified by pcr using primers b4343 and b4344 ( fig4 ). the resulting pcr product may then be cleaned , digested with ncoi and paci , and cleaned again . the two pcr products may then be ligated , in a 3 - way ligation , to psmx309 that has been digested with kpni and paci and cleaned prior to ligation , to yield plasmid psmx310 ( fig4 ). primer b4341 consists of a 5 ′ kpni restriction site ( underlined ), followed by a bi - directional transcription terminator ( tonb terminator ), followed by sequence complementary to the end of sasp - c from bacillus megaterium strain km ( atcc 13632 ) ( underlined , in bold ; fig4 ). primer b4342 ( fig4 ) consists of a 5 ′ ncoi restriction site ( underlined ), followed by sequence of the beginning of the sasp - c gene from bacillus megaterium strain km ( atcc 13632 ). primer b4343 consists of a 5 ′ ncoi restriction site ( underlined ), followed by sequence of the fda promoter ( fig4 ). primer b4344 consists of a 5 ′ paci restriction site ( underlined ), followed by sequence complementary to the fda promoter ( fig4 ). genetic modification of phi33 to simultaneously delete the phi33 endolysin gene , and add fda - sasp - c to the bacteriophage genome , utilising the ptp92 host range determinant as a means of selection 1 . plasmid psmx310 ( fig4 ) may be introduced into a p . aeruginosa strain that is a host for both the original , and the host range determinant donor phage , by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / m1 ), yielding strain pta33 . 2 . strain pta33 may be infected with phage phi33 , and the progeny phage harvested . 3 . recombinant phage , in which the ptp92 host range determinant has been transferred to phi33 , may be identified by plaquing the lysate from step ( 2 ) on p . aeruginosa strain pax31 ( endolysin +; fig1 ), i . e . a strain 2726 derivative , which is a host for the recombinant phage that carries the ptp92 host range determinant , but which is not a host for the parental bacteriophage phi33 , and which carries the phi33 endolysin gene in trans . 4 . a pcr screen may be further carried out to identify isolates that have simultaneously acguired fda - sasp - c , in addition to the host range determinant from ptp92 . 5 . isolates may further be tested for the endolysin deletion by plaquing on unmodified p . aeruginosa strain 2726 ( endolysin ), as phage isolates from which the endolysin has been successfully removed will fail to plaque on this strain . 5 . following identification of a verified isolate ( ptpx33 ; fig5 ), this isolate may be plaque purified twice more on an endolysin + p . aeruginosa strain , prior to further use . strains were tested for sensitivity to each phage by dropping 10 μl of crude phage lysate onto a soft agar overlay plate inoculated with bacteria . plates were grown overnight at 32 ° c . and the strains were scored for sensitivity to each phage by assessing clearance zones at the point of inoculation . where phage inhibited growth , as seen by clearance of the bacterial lawn , the strain was marked as sensitive (+), and where no inhibition of growth was seen , the strain was marked as not - sensitive (−) abedon s t . 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