Patent Application: US-201515517656-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:
the invention will now be described in further detail , by way of example only , with reference to the accompanying figures and the following examples . fig1 is a schematic diagram showing construction of plasmids containing laczδm15 and the phi33 endolysin gene for the creation of transgenic p . aeruginosa strains ; fig2 is a schematic diagram showing construction of plasmids encoding hybrid tail fibre genes , including the laczα marker ; fig3 is a schematic diagram showing construction of plasmids encoding hybrid tail fibre genes , which do not include the laczα marker ; fig4 is a schematic diagram showing construction of phage with hybrid tail fibre genes ; fig5 is a schematic diagram showing construction of plasmids for the genetic modification of phage to introduce an additional tail fibre gene or tail fibre hybrid gene , utilising a laczα marker ; fig6 is a schematic diagram showing genetic modification of phage to add an extra tail fibre gene , utilising a laczα marker , and then to replace endolysin with rpsb - sasp - c , also utilising a laczα marker ; fig7 is a schematic diagram showing genetic modification of further phage to add an extra tail fibre hybrid gene , utilising a laczα marker , and then to replace endolysin with rpsb - sasp - c , also utilising a laczα marker ; fig8 is a schematic diagram showing genetic modification of further phage to add an extra tail fibre hybrid gene , utilising a laczα marker , and then to replace endolysin with rpsb - sasp - c , also utilising a laczα marker ; fig9 is a schematic diagram showing construction of plasmids for the genetic modification of phage to add a third tail fibre hybrid gene , utilising a laczα marker ; fig1 is a schematic diagram showing genetic modification of phage carrying three tail fibre genes , utilising a laczα marker , and then to replace endolysin with rpsb - sasp - c , also utilising a laczα marker ; fig1 is a schematic diagram showing construction of plasmids for the genetic modification production of phage to replace the endolysin gene with rpsb - sasp - c , utilising a laczα marker ; fig1 is a schematic diagram showing production of bacteriophage that carry multiple tail fibre genes or tail fibre hybrid genes , and in which the endolysin gene has been replaced with rpsb - sasp - c , according to the invention ; fig1 is a schematic diagram showing production of further bacteriophage that carry multiple tail fibre genes or tail fibre hybrid genes , and in which the endolysin gene has been replaced with rpsb - sasp - c , according to the invention ; and fig1 is a multiple sequence alignment of tail fibre genes from related phages . generic product covering multiple tail fibres within an individual phage , or a mix of phages each containing multiple tail fibres summary of the genetic modification of a lytic bacteriophage to render it non - lytic , and such that it carries more than one tail fibre gene , 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 phi33 derivatives , it is shown how , using an e . coli / p . aeruginosa broad host range vector , as an example only , phi33 - based bacteriophage carrying alternative tail fibre genes may be made , via homologous recombination . it is also shown how phi33 derivatives may be constructed , using an e . coli / p . aeruginosa broad host range vector , as an example only , in which an additional tail fibre gene is added to the bacteriophage genome via homologous recombination , such that the resulting bacteriophage carry two tail fibre genes . in a subsequent step , it is shown how , using an e . coli / p . aeruginosa broad host range vector , as an example only , a third tail fibre gene may be added to the bacteriophage genome via homologous recombination , such that the resulting bacteriophage carry three tail fibre genes . as an example , for the construction of recombinant lytic bacteriophage , an e . coli laczα marker may be included as a means of identifying recombinant bacteriophage . in order to use this marker , the bacteriophage host strains must first be modified to carry the e . coli laczδm15 allele at a suitable location in the bacterial genome , to complement the laczα phenotypes of the desired recombinant bacteriophage . as an example , the construction of this p . aeruginosa strain 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 laczδm15 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 . 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 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 acquisition of tetracycline ( 50 μg / ml ) resistance . isolates which have undergone a second homologous recombination event may then be isolated on medium containing 10 % sucrose ( utilising the sacb counterselectable marker that is present on the plasmid backbone ). as an example by which phi33 derivatives may be made that possess an alternative tail fibre gene , a tail fibre gene comprising the region encoding the n - terminal region of the phi33 tail fibre , followed by the region encoding the c - terminal , receptor - binding region of the tail fibre from phage ptp92 ( phi33 ( n ) ptp92 ( c )), may be constructed , and cloned next to a laczα marker , in between two regions of homology that flank the native tail fibre gene of phi33 . this plasmid may be introduced into p . aeruginosa , and the resulting strain infected with phi33 . following harvesting of progeny phage , double recombinants may be isolated by plaquing on a suitable p . aeruginosa ( laczδm15 + ) host , using medium containing s - gal as a chromogenic indicator of β - galactosidase activity . the resulting phage will have had the native phi33 tail fibre replaced by the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre , plus a laczα marker . in a subsequent step , the laczα marker may be removed from the phi33 ( n ) ptp92 ( c ) tail fibre phage via another homologous recombination step . the region of homology downstream of the native phi33 tail fibre may be cloned next to the gene encoding the c - terminal , receptor - binding region of ptp92 . this plasmid may be introduced into a suitable p . aeruginosa strain , and the resulting strain infected with the phi33 derivative carrying the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre , plus laczα . following harvesting of progeny phage , double recombinants may be isolated by plaquing on a suitable p . aeruginosa ( laczδm15 + ) host , using medium containing s - gal as a chromogenic indicator of β - galactosidase activity . the resulting phi33 derivative ( ptp93 ) will have had the native phi33 tail fibre replaced by the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre , and will no longer carry the laczα marker . as an example by which tail fibre genes may be added to a bacteriophage genome , the tail fibre gene from bacteriophage phi33 may be cloned next to the e . coli laczα marker , between two regions of phi33 dna that flank the 5 ′ end of orf57 ( ectopic position 1 ; this is the beginning of the predicted operon containing the native tail fibre gene ), in a broad host range e . coli / p . aeruginosa vector . this plasmid may be introduced into p . aeruginosa , and the resulting strain infected with ptp93 . following harvesting of progeny phage , double recombinants may be isolated by plaquing on a suitable p . aeruginosa ( laczδm15 + ) host , using medium containing s - gal as a chromogenic indicator of β - galactosidase activity . the resulting phage will contain two tail fibre genes : the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native position and the gene encoding the native phi33 tail fibre ( plus a laczα marker ) at an ectopic position ( ectopic position 1 ). in an alternative example , a gene encoding a tail fibre comprising the n - terminal region of the phi33 tail fibre and the c - terminal receptor - binding region of the tail fibre from bacteriophage ptp47 ( phi33 ( n ) ptp47 ( c )), may be constructed and cloned next to the e . coli laczα marker , between two regions of phi33 dna that flank the 5 ′ end of orf57 ( ectopic position 1 ; this is the beginning of the predicted operon containing the native tail fibre gene ), in a broad host range e . coli / p . aeruginosa vector . this plasmid may be introduced into p . aeruginosa , and the resulting strain infected with ptp93 . following harvesting of progeny phage , double recombinants may be isolated by plaquing on a suitable p . aeruginosa ( laczδm15 + ) host , using medium containing s - gal as a chromogenic indicator of β - galactosidase activity . the resulting phage will contain two tail fibre genes : the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native position and the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre ( plus a laczα marker ) at an ectopic position ( ectopic position 1 ). in an alternative example , a gene encoding a tail fibre comprising the n - terminal region of the phi33 tail fibre and the c - terminal receptor - binding region of the tail fibre from bacteriophage ptp47 ( phi33 ( n ) ptp47 ( c )), may be constructed and cloned next to an e . coli laczα marker , between two regions of phi33 dna that flank orf57 ( ectopic position 1 ; this is the beginning of the predicted operon containing the native tail fibre gene ), 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 may be isolated by plaquing on a suitable p . aeruginosa ( laczδm15 + ) host , using medium containing s - gal as a chromogenic indicator of β - galactosidase activity . the resulting phage will contain two tail fibre genes : the gene encoding the phi33 native tail fibre at the native position and the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre ( plus a laczα marker ) at an ectopic position ( ectopic position 1 ). in subsequent steps , the laczα marker may be removed from these phi33 derivatives by another homologous recombination step . the laczα marker may be deleted from the previously - described recombination plasmids that were used to introduce the gene encoding the phi33 native tail fibre , or the phi33 ( n ) ptp47 ( c ) tail fibre at ectopic position 1 . these δlaczα plasmids may be introduced into suitable p . aeruginosa strains , and the resulting strains infected , as appropriate , with phi33 derivatives carrying either the wild type phi33 tail fibre gene plus the laczα marker , or the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre plus the laczα marker , at ectopic position 1 . following harvesting of progeny phage , double recombinants may be isolated by plaquing on a suitable p . aeruginosa ( laczδm15 + ) host , using medium containing s - gal as a chromogenic indicator of β - galactosidase activity . the resulting phi33 derivatives will contain two tail fibre genes ( phi33 ( n ) ptp92 ( c ) at the native position and phi33 native tail fibre at ectopic position 1 , or phi33 ( n ) ptp92 ( c ) at the native position and phi33 ( n ) ptp47 ( c ) at ectopic position 1 , or phi33 native tail fibre at the native position and phi33 ( n ) ptp47 ( c ) at ectopic position 1 ), and will no longer carry the laczα marker . in a subsequent step , another homologous recombination may be used to add a third tail fibre gene to the bacteriophage genome . as an example , a gene encoding a tail fibre comprising the n - terminal region of the phi33 tail fibre and the c - terminal receptor - binding region of the tail fibre from bacteriophage ptp47 , under the control of the native tail fibre promoter ( orf57 promoter ), may be constructed and cloned next to a laczα marker , between two regions of phi33 dna that flank an intergenic region between orf28 and orf29 ( ectopic position 2 ), 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 carrying the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native position and the gene encoding the native phi33 tail fibre at ectopic position 1 ( δlaczα ). following harvesting of progeny phage , double recombinants may be isolated by plaquing on a suitable p . aeruginosa ( laczδm15 + ) host , using medium containing s - gal as a chromogenic indicator of β - galactosidase activity . the resulting phage will contain three tail fibre genes : the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native position , the native phi33 tail fibre gene at ectopic position 1 , and the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre ( plus a laczα marker ) at ectopic position 2 . in a subsequent step , the laczα marker may be removed from this phi33 derivative carrying three tail fibre genes ( the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native locus , the native phi33 tail fibre gene at ectopic position 1 , and the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre , plus the laczα marker , at ectopic position 2 ) by another homologous recombination step . the laczα marker may be deleted from the previously - described recombination plasmid used to introduce the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre at ectopic position 2 . this δlaczα plasmid may be introduced into a suitable p . aeruginosa strain , and the resulting strain infected with the phi33 derivative carrying the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native locus , the native phi33 tail fibre gene at ectopic position 1 , and the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre , plus the laczα marker , at ectopic position 2 . following harvesting of progeny phage , double recombinants may be isolated by plaquing on a suitable p . aeruginosa ( laczδm15 + ) host , using medium containing s - gal as a chromogenic indicator of β - galactosidase activity . the resulting phage will contain three tail fibre genes : the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native position , the native phi33 tail fibre gene at ectopic position 1 , and the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre at ectopic position 2 , and will no longer carry the laczα marker . in subsequent steps , a similar homologous recombination may be used to replace the endolysin gene of any of the phi33 derivatives , or similar bacteriophage , with the gene for sasp - c , under the control of a p . aeruginosa rpsb promoter , while simultaneously adding the e . coli laczα marker for the identification of recombinant phage . since the bacteriophage to be modified is lytic ( rather than temperate ), another requirement for this latter step of bacteriophage construction is the construction of a derivative of a p . aeruginosa host strain that carries the phi33 endolysin gene and the e . coli laczδm15 allele at a suitable location in the bacterial genome , to complement the δendolysin and laczα phenotypes of the desired recombinant bacteriophage . as an example , the construction of this p . aeruginosa strain 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 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 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 acquisition of tetracycline ( 50 μg / ml ) resistance . isolates which have undergone a second homologous recombination event may then be isolated on medium containing 10 % sucrose ( utilising the sacb counter - selectable marker that is present on the plasmid backbone ). 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 the previously constructed p . aeruginosa ( endolysin + laczδm15 + ) strain , and the resulting strain infected by any of the phi33 derivatives that have already been genetically modified to carry more than one tail fibre gene , as exemplified above in the previous steps . progeny phage may be harvested and double recombinants identified by plaquing on p . aeruginosa ( endolysin + laczδm15 + ), looking for acquisition of the laczα reporter on medium containing a chromogenic substrate that detects the action of β - galactosidase . in a subsequent step , the laczα marker may be removed from the previously - constructed phage that carry rpsb - sasp - c and laczα in place of the endolysin gene , by homologous recombination . a region consisting of rpsb - sasp - c may be cloned in between two regions of homology that flank the phi33 endolysin gene , in a broad host range e . coli / p . aeruginosa vector . this plasmid may be transferred to the previously constructed p . aeruginosa ( endolysin + laczδm15 + ) strain , and the resulting strain infected by any of the phi33 derivatives that have already been genetically modified to carry rpsb - sasp - c in place of the endolysin gene , as exemplified above in the previous steps . 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 . the resulting phage will carry multiple tail fibre or tail fibre hybrid genes , and carry rpsb - sasp - c in place of endolysin , according to the invention . 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 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 . 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 . 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 a plasmid to introduce the escherichia coli laczδm15 allele into the genome of p . aeruginosa , downstream of phoa 1 . plasmid psmx400 ( 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 b4400 and b4401 ( fig1 ). the pcr product may then be cleaned and digested with spel 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 b4402 and b4403 ( fig1 ). this second pcr product may then be cleaned and digested with bglii and xhol . the two digests may be cleaned again and ligated to psm1104 that has been digested with spel and xhoi , in a 3 - way ligation , to yield plasmid psmx400 ( fig1 ). primer b4400 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 b4401 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 b4402 consists of 5 ′ bglii and nhel restriction sites ( underlined ), followed by sequence immediately downstream of the stop codon of the phoa gene from p . aeruginosa strain pao1 ( fig1 ). primer b4403 consists of a 5 ′ xhol 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 psmx401 ( fig1 ), comprising psmx400 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 b4408 and b4409 ( fig1 ). the resulting pcr product may then be digested with bglii and nhel , and ligated to psmx400 that has also been digested with bglii and nhel , to yield plasmid psmx401 ( fig1 ). primer b4408 consists of a 5 ′ bglii restriction site ( underlined ), followed by sequence of the lac promoter ( fig1 ). primer b4409 consists of a 5 ′ nhel 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 escherichia coli laczδm15 gene immediately downstream of the phoa locus of the bacterial genome 1 . plasmid psmx401 ( 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 counter - selection , 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 ( pax40 ), this strain may then be used as a host for further modification of bacteriophage , where complementation of a laczα reporter is required . construction of plasmids for recombination with phi33 , to generate ptp93 , utilising a laczα screening process 1 . psmx402 ( 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 b4422 and b4449 ( fig2 ). the resulting pcr product may then be cleaned and digested with nhel , and ligated to psm1080 that has also been digested with nhel and then treated with alkaline phosphatase prior to ligation , yielding plasmid psmx402 ( fig2 ). primer b4422 consists of a 5 ′ nhel restriction site ( underlined ), followed by sequence from phi33 , approximately 1 kb downstream of the end of the phi33 tail fibre gene ( fig2 ). b4449 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 ). 2 . psmx403 ( fig2 ), comprising psmx402 carrying laczα , a tail fibre gene consisting of a 3 ′ section of ptp92 dna that encodes the c - terminal receptor - binding region of the tail fibre and the 5 ′ section of the phi33 tail fibre gene sequence that encodes the n - terminal region , 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 b4450 and b4452 ( fig2 ). the region of the ptp92 tail fibre gene that encodes the c - terminal receptor - binding region , may be amplified by pcr from ptp92 using primers b4451 and b4454 ( fig2 ). the laczα open reading frame may then be joined to the section of ptp92 dna that encodes the tail fibre c - terminal receptor - binding region , by soeing pcr using the outer primers , b4450 and b4454 . a region comprising sequence of phi33 tail fibre gene that encodes the n - terminal region , and sequence located immediately upstream of the phi33 tail fibre gene , may be amplified by pcr using primers b4453 and b4429 ( fig2 ). this pcr product may then be joined to the pcr product comprising laczα and the ptp92 tail fibre gene section , by soeing pcr using the outer primers b4450 and b4429 . the resulting pcr product may then be cleaned and digested with avrii and kpni , and ligated to psmx402 that has also been digested with avrii and kpni , yielding plasmid psmx403 ( fig2 ). primer b4450 consists of a 5 ′ avrii restriction site , followed by sequence complementary to the 3 ′ end of the laczα open reading frame ( fig2 ). primer b4452 consists of a 5 ′ section of sequence that overlaps the 3 ′ end ptp92 tail fibre region that encodes the c - terminal receptor - binding region , followed by sequence of the 5 ′ end of the laczα open reading frame ( fig2 ). primer b4451 is the reverse complement of primer b4452 ( fig2 ). primer b4454 consists of 5 ′ sequence from within the region of the phi33 tail fibre gene that encodes the n - terminal region ( underlined ), followed sequence within the region of the ptp92 tail fibre gene that encodes the c - terminal receptor - binding region ( fig2 ). primer b4453 is the reverse complement of primer b4454 . primer b4429 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 ). 3 . psmx404 ( fig3 ), comprising psm1080 carrying a region of the gene encoding the c - terminal receptor - binding region of the ptp92 tail fibre , 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 b4422 and b4455 ( fig3 ). the region of the gene encoding the c - terminal receptor - binding region of the ptp92 tail fibre may be amplified by pcr using primers b4456 and b4457 ( fig3 ). these two pcr products may then be joined by soeing pcr , using the two outer primers b4422 and b4457 . the resulting pcr product may then be cleaned , digested with nhel , cleaned again , and ligated to psm1080 that has also been digested with nhel and then treated with alkaline phosphatase prior to ligation , to yield plasmid psmx404 ( fig3 ). primer b4455 consists of a 5 ′ section of the region of the gene encoding the c - terminal receptor - binding region of the ptp92 tail fibre gene ( underlined ), followed by sequence immediately downstream of the phi33 tail fibre gene ( fig3 ). primer b4456 is the reverse complement of primer b4455 ( fig3 ). primer b4457 consists of a 5 ′ nhel restriction site ( underlined ), followed by sequence of a region within the section of the tail fibre gene of ptp92 , that encodes the c - terminal , receptor - binding region ( fig3 ). genetic modification of phi33 to replace the 3 ′ region of the tail fibre gene , encoding the c - terminal receptor - binding region , with that of ptp92 , to form the phi33 ( c ) ptp92 ( n ) tail fibre gene , at the native position within the phi33 genome 1 . plasmid psmx403 ( fig2 ; fig4 ) may be introduced into p . aeruginosa strain pax40 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta40 . 2 . strain pta40 may be infected with phage phi33 , and the progeny phage harvested . 3 . recombinant phage in which the region of the phi33 gene encoding the c - terminal , receptor - binding region of the tail fibre 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 pax40 , 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 ( ptpx40 ; fig4 ), this isolate may be plaque purified twice more on p . aeruginosa strain pax40 , prior to further use . genetic modification of ptpx40 to remove the laczα marker , generating ptp93 ( phi33 , carrying the phi33 ( n ) ptp92 ( c ) tail fibre gene ) 1 . plasmid psmx404 ( fig3 ; fig4 ) may be introduced into p . aeruginosa strain pax40 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta41 . 2 . strain pta41 may be infected with phage ptpx40 , 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 pax40 , onto medium containing s - gal , looking for white plaques , which is 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 ( ptp93 ; fig4 ), this isolate may be plaque purified twice more on p . aeruginosa strain pax40 , prior to further use . construction of plasmids for the genetic modification of ptp93 to introduce either the phi33 tail fibre gene , or the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre at ectopic position 1 1 . plasmid psmx405 ( fig5 ), comprising psm1080 containing a 2 . 8 kb fragment of phi33 spanning a continuous ‘ orf60 to orf57 ’ stretch ( neither orf60 nor orf57 were complete ), may be constructed as follows . fig5 shows the priming sites for the oligonucleotides described below for amplification of regions from the phi33 genome . pcr amplification of phi33 dna may be carried out using primers b4410 and b3332 ( fig5 ), to yield a 2 . 9 kb fragment , which may be cleaned and digested with pcii . following digestion , the dna may be cleaned and ligated to psm1080 that has been digested with ncol and treated with alkaline phosphatase , to yield psmx405 ( fig5 ). primer b4410 consists of a 5 ′ pcil site ( underlined ; fig5 ), followed by sequence that anneals approximately 240 bp downstream of the start codon of phi33 orf60 , in the sense orientation . primer b3332 consists of sequence complementary to phi33 orf57 , and anneals approximately 120 bp downstream of an intrinsic pcii site within orf57 . 2 . plasmid psmx406 ( fig5 ), comprising psmx405 carrying the complete tail fibre gene from phi33 and a promoterless laczα marker , may be constructed as follows . the complete tail fibre gene from phi33 ( fig5 ) may be amplified by pcr using primers b3324 and b4411 . a promoterless laczα marker may be amplified by pcr from puc19 using primers b4412 and b4413 ( fig5 ). the two pcr products may be joined together by soeing pcr using the two outer primers , b3324 and b4413 . the resulting pcr product may then be digested with bstbi , and ligated to psmx405 that has also been digested with bstbi and treated with alkaline phosphatase prior to ligation . plasmid psmx406 may be isolated following screening of clones to identify a clone in which the phi33 tail fibre has been cloned in the same orientation as orf57 ( fig5 ). primer b3324 consists of a 5 ′ bstbi site ( underlined ), followed by sequence that anneals to the ribosome binding site just upstream of the phi33 tail fibre gene ( fig5 ). primer b4411 consists of 5 ′ sequence complementary to the beginning of the laczα marker from puc19 , followed by sequence complementary to the end of the tail fibre gene from phi33 ( underlined ; fig5 ). primer b4412 is the reverse complement of primer b4411 ( fig5 ). primer b4413 consists of a 5 ′ bstbi site ( underlined ), followed by sequence complementary to the region between the native phi33 bstbi site and orf57 , followed in turn by sequence complementary to the end of the laczα marker from puc19 ( fig5 ). 3 . plasmid psmx407 ( fig5 ), comprising psmx405 carrying a tail fibre gene consisting of the 5 ′ section of phi33 dna encoding the n - terminal region of the phi33 tail fibre , and the 3 ′ section of ptp47 dna encoding the c - terminal , receptor - binding region of the ptp47 tail fibre , in addition to a promoterless laczα marker , may be constructed as follows . the dna region encoding the n - terminal region of the phi33 tail fibre may be amplified by pcr using primers b3324 and b4417 ( fig5 ). the dna region encoding the c - terminal , receptor - binding region of the ptp47 tail fibre may be amplified by pcr using primers b4416 and b4414 ( fig5 ). the two pcr products may be joined together by soeing pcr using the outer primers b3324 and b4414 . the laczα marker from puc19 may be amplified by pcr using primers b4415 and b4413 ( fig5 ). the laczα marker may be joined to the constructed tail fibre pcr product by soeing pcr using the outer primers b3324 and b4413 . the resulting pcr product may then be digested with bstbi and ligated to psmx405 that has been digested with bstbi and treated with alkaline phosphatase prior to ligation . plasmid psmx407 may be isolated following screening of clones to identify a clone in which the phi33 ( n ) ptp47 ( c ) tail fibre has been cloned in the same orientation as orf57 ( fig5 ). primer b4417 consists of a 5 ′ section of sequence complementary to part of ptp47 encoding the c - terminal , receptor - binding region of the ptp47 tail fibre ( underlined ), followed by sequence complementary to part of phi33 encoding the n - terminal region of the phi33 tail fibre ( fig5 ). primer b4416 is the reverse complement of b4417 ( fig5 ). b4414 consists of 5 ′ sequence complementary to the beginning of the laczα marker from puc19 , followed by sequence complementary to the end of the tail fibre gene from ptp47 ( underlined ; fig5 ). primer b4415 is the reverse complement of primer b4414 ( fig5 ). genetic modification of ptp93 to add the phi33 tail fibre gene and a laczα marker , upstream of orf57 1 . psmx406 ( fig5 ; fig6 ) may be introduced into p . aeruginosa strain pml14 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta42 . 2 . strain pta42 may be infected with ptp93 , and the progeny phage harvested . 3 . recombinant phage , which have acquired the phi33 tail fibre and laczα marker upstream of orf57 , may be identified by plaquing on p . aeruginosa strain pax40 using medium containing s - gal , a chromogenic substrate that detects β - galactosidase activity , looking for black plaques . 4 . pcr may be carried out to confirm that the phi33 tail fibre and laczα marker have been introduced upstream of orf57 in ptp93 , and to confirm that the native ptp93 tail fibre region is still intact . 5 . following identification of a verified isolate ( ptpx41 ; fig6 ), the new recombinant phage may be plaque purified twice more on p . aeruginosa strain pax40 , before further use . ptpx41 is therefore a phi33 derivative carrying the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native position , and the phi33 wild type tail fibre gene along with a laczα marker at ectopic position 1 . genetic modification of ptp93 to add the phi33 ( n ) ptp47 ( c ) tail fibre gene and a laczα marker , upstream of orf57 1 . psmx407 ( fig5 ; fig7 ) may be introduced into p . aeruginosa strain pml14 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta43 . 2 . strain pta43 may be infected with ptp93 , and the progeny phage harvested . 3 . recombinant phage , which have acquired the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre , in addition to the laczα marker , upstream of orf57 , may be identified by plaquing on p . aeruginosa strain pax40 using medium containing s - gal , a chromogenic substrate that detects β - galactosidase activity , looking for black plaques . 4 . pcr may be carried out to confirm that the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre , in addition to the laczα marker , has been introduced upstream of orf57 in ptp93 , and to confirm that the native ptp93 tail fibre region is still intact . 5 . following identification of a verified isolate ( ptpx42 ; fig7 ), the new recombinant phage may be plaque purified twice more on p . aeruginosa strain pax40 , before further use . ptpx42 is therefore a phi33 derivative carrying the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native position , and the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre along with a laczα marker at ectopic position 1 . genetic modification of phi33 to add the phi33 ( n ) ptp47 ( c ) tail fibre gene and a laczα marker , upstream of orf57 1 . psmx407 ( fig5 ; fig8 ) may be introduced into p . aeruginosa strain pml14 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta43 . 2 . strain pta43 may be infected with phi33 , and the progeny phage harvested . 3 . recombinant phage , which have acquired the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre , in addition to the laczα marker , upstream of orf57 , may be identified by plaquing on p . aeruginosa strain pax40 using medium containing s - gal , a chromogenic substrate that detects β - galactosidase activity , looking for black plaques . 4 . pcr may be carried out to confirm that the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre , in addition to the laczα marker , has been introduced upstream of orf57 in phi33 , and to confirm that the native phi33 tail fibre region is still intact . 5 . following identification of a verified isolate ( ptpx43 ; fig8 ), the new recombinant phage may be plaque purified twice more on p . aeruginosa strain pax40 , before further use . ptpx43 is therefore a phi33 derivative carrying the native phi33 tail fibre gene at the native position , and the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre along with a laczα marker at ectopic position 1 . construction of plasmids to remove the laczα markers from the double - tail fibre phage , ptpx41 , ptpx42 and ptpx43 1 . plasmid psmx408 ( fig5 ), consisting of psmx405 carrying the phi33 tail fibre gene , may be constructed as follows . the phi33 tail fibre gene may be amplified by pcr using primers b3324 and b3333 ( fig5 ). the resulting pcr product may then be digested with bstbi and ligated to psm405 that has been digested with bstbi and treated with alkaline phosphatase prior to ligation , to yield plasmid psmx408 . primer b3333 consists of a 5 ′ bstbi site ( underlined ), followed by sequence complementary to the region between the native phi33 bstbi site and orf57 , followed in turn by sequence complementary to the 3 ′ end of the tail fibre gene from phi33 ( fig5 ). 2 . plasmid psmx409 ( fig5 ), consisting of psmx405 carrying the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre , may be constructed as follows . the gene encoding the phi33 ( n ) ptp74 ( c ) tail fibre may be amplified by pcr from psmx407 using primers b3324 and b4418 ( fig5 ). the resulting pcr product may then be digested with bstbi and ligated to psm405 that has been digested with bstbi and treated with alkaline phosphatase prior to ligation , to yield plasmid psmx409 ( fig5 ). primer b4418 consists of a 5 ′ bstbi site ( underlined ), followed by sequence complementary to the region between the native phi33 bstbi site and orf57 , followed in turn by sequence complementary to the 3 ′ end of the tail fibre gene from ptp47 ( fig5 ). 1 . psmx408 ( fig5 ; fig6 ) may be introduced into p . aeruginosa strain pml14 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta44 . 2 . strain pta44 may be infected with ptpx41 , and the progeny phage harvested . 3 . recombinant phage , from which the laczα marker has been removed , may be identified by plaquing on p . aeruginosa strain pax40 using medium containing s - gal , a chromogenic substrate that detects β - galactosidase activity , looking for clear plaques . 4 . pcr may be carried out to confirm that the laczα marker has been removed , and that the two tail fibre genes are still intact . 5 . following identification of a verified isolate ( ptpx44 ; fig6 ), the new recombinant phage may be plaque purified twice more on p . aeruginosa strain pax40 , before further use . ptpx44 is therefore a phi33 derivative carrying the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native position , and the phi33 tail fibre gene at ectopic position 1 ( alaczα ). removal of laczα marker from the double - tail fibre phage ptpx42 and ptpx43 1 . psmx409 ( fig5 ; fig7 ; fig8 ) may be introduced into p . aeruginosa strain pml14 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta45 . 2 . strain pta45 may be infected with ptpx42 ( fig7 ) or ptpx43 ( fig8 ), as appropriate , and the progeny phage harvested . 3 . recombinant phage , from which the laczα marker has been removed , may be identified by plaquing on p . aeruginosa strain pax40 using medium containing s - gal , a chromogenic substrate that detects β - galactosidase activity , looking for clear plaques . 4 . pcr may be carried out to confirm that the laczα marker has been removed , and that the two tail fibre genes are still intact . 5 . following identification of verified isolates , the new recombinant phage may be plaque purified twice more on p . aeruginosa strain pax40 , before further use . ptpx45 ( fig7 ) is therefore a phi33 derivative carrying the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native position and the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre at ectopic position 1 ( alaczα ). ptpx46 ( fig8 ) is therefore a phi33 derivative carrying the phi33 tail fibre gene at the native position and the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre at ectopic position 1 ( alaczα ). construction of a plasmid to add a third tail fibre gene to ptpx44 , at ectopic position 2 , in the intergenic region between orf28 and orf29 1 . plasmid psmx410 ( fig9 ), comprising psm1080 carrying sequences of phi33 dna that flank the orf28 - 29 intergenic region , may be constructed as follows . a region of phi33 dna flanking the end of orf28 may be amplified by pcr using primers b4419 and b4420 ( fig9 ). a region of phi33 dna flanking the beginning of orf29 may be amplified by pcr using primers b4421 and b4422 ( fig9 ). the resulting pcr products may then be joined by soeing pcr using the outer primers , b4419 and b4422 . the joined pcr product may be cleaned , digested with nhel and ligated to psm1080 that has been digested with nhel and treated with alkaline phosphatase prior to ligation , to yield plasmid psmx410 ( fig9 ). primer b4419 consists of a 5 ′ nhel restriction site ( underlined ), followed by phi33 sequence within orf28 ( fig9 ). primer b4420 consists of 5 ′ sequence complementary to that of the phi33 orf28 - orf29 intergenic region , kpni and aflii restriction sites ( underlined ), followed by sequence complementary to more of the phi33 orf28 - orf29 intergenic region ( fig9 ). primer b4421 is the reverse complement of primer b4420 ( fig9 ). primer b4422 consists of a 5 ′ nhel restriction site ( underlined ), followed by phi33 sequence complementary to the region downstream of orf29 ( fig9 ). 2 . plasmid psmx411 ( fig9 ), comprising psmx410 carrying a gene constructed to encode the phi33 ( n ) ptp47 ( c ) tail fibre , under the control of the native tail fibre promoter ( porf57 ), in addition to a laczα marker , may be constructed as follows . the dna region comprising [ porf57 - phi33 ( n ) ptp47 ( c ) tail fibre gene - laczα ] may be amplified from plasmid psmx407 ( fig5 ), by pcr using primers b4423 and b4424 ( fig9 ). the resulting pcr product may be digested with aflii and kpni , and ligated to psmx410 that has also been digested with aflii and kpni , to yield plasmid psmx411 ( fig9 ). primer b4423 consists of a 5 ′ aflii restriction site ( underlined ), followed by sequence of the phi33 orf57 promoter ( fig9 ). primer b4424 consists of a 5 ′ kpni restriction site ( underlined ), followed by sequence that is complementary to the end of the laczα marker ( fig9 ). genetic modification of ptpx44 to add the phi33 ( n ) ptp47 ( c ) tail fibre gene and a laczα marker , in the intergenic region between orf28 and orf29 ( ectopic position 2 ), to generate a bacteriophage carrying three tail fibre genes 1 . psmx411 ( fig9 ; fig1 ) may be introduced into p . aeruginosa strain pml14 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta46 . 2 . strain pta46 may be infected with ptpx44 , and the progeny phage harvested . 3 . recombinant phage , which have acquired the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre , in addition to the laczα marker , in the orf28 - 29 intergenic region , may be identified by plaquing on p . aeruginosa strain pax40 using medium containing s - gal , a chromogenic substrate that detects β - galactosidase activity , looking for black plaques . 4 . pcr may be carried out to confirm that the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre , in addition to the laczα marker , has been introduced into the orf28 - 29 intergenic region and to confirm the presence of the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native position , and the native phi33 tail fibre gene at ectopic position 1 . 5 . following identification of a verified isolate ( ptpx47 ; fig1 ), the new recombinant phage may be plaque purified twice more on p . aeruginosa strain pax40 , before further use . ptpx47 is therefore a phi33 derivative carrying the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native position , the native phi33 tail fibre gene at ectopic position 1 , and the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre along with a laczα marker at ectopic position 2 . construction of a plasmid to remove the laczα marker from the triple - tail fibre bacteriophage , ptpx47 1 . plasmid psmx412 ( fig9 ), comprising psmx410 carrying the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre , under the control of the native promoter ( porf57 ) may be constructed as follows . the [ porf57 - phi33 ( n ) ptp47 ( c ) tail fibre gene ] region from psmx407 ( fig5 ) may be amplified by pcr using primers b4423 and b4425 ( fig9 ). the resulting pcr product may be digested with aflii and kpni and ligated to psmx410 that has also been digested with aflii and kpni , to yield plasmid psmx412 ( fig9 ). primer b4425 consists of a 5 ′ kpni site ( underlined ), followed by sequence complementary to the end of the ptp47 tail fibre gene ( fig9 ). genetic modification of the triple - tail fibre bacteriophage , ptpx47 to remove the laczα marker 1 . psmx412 ( fig9 ; fig1 ) may be introduced into p . aeruginosa strain pml14 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta47 . 2 . strain pta47 may be infected with ptpx47 , and the progeny phage harvested . 3 . recombinant phage , from which the laczα marker has been removed , may be identified by plaquing on p . aeruginosa strain pax40 using medium containing s - gal , a chromogenic substrate that detects β - galactosidase activity , looking for clear plaques , indicative of loss of β - galactosidase activity . 4 . pcr may be carried out to confirm that the laczα marker has been removed , and that the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre is still present in the orf28 - 29 intergenic region ( ectopic position 2 ), and to confirm the presence of the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native position , and the native phi33 tail fibre gene at ectopic position 1 . 5 . following identification of a verified isolate ( ptpx48 ; fig1 ), the new recombinant phage may be plaque purified twice more on p . aeruginosa strain pax40 , before further use . ptpx48 is therefore a phi33 derivative carrying the gene encoding the phi33 ( n ) ptp92 ( c ) tail fibre at the native position , the native phi33 tail fibre gene at ectopic position 1 , and the gene encoding the phi33 ( n ) ptp47 ( c ) tail fibre ( alaczα ) at ectopic position 2 . construction of a plasmid to generate a p . aeruginosa strain carrying the phi33 endolysin gene and the escherichia coli laczδm15 immediately downstream of the phoa locus of the bacterial genome 1 . plasmid psmx413 ( fig1 ), comprising psmx400 carrying the endolysin gene from phi33 , under the control of the native endolysin promoter , may be constructed as follows . the endolysin promoter may be amplified by pcr from phi33 using primers b4404 and b4405 ( fig1 ). the endolysin gene itself may be amplified by pcr from phi33 using primers b4406 and b4407 ( fig1 ). the two pcr products may then be joined together by splicing by overlap extension ( soeing ) pcr , using the two outer primers , b4404 and b4407 . the resulting pcr product may then be digested with aflii and bglii , and ligated to psmx400 that has also been digested with aflii and bglii , to yield plasmid psmx413 ( fig1 ). primer b4404 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 endolysin promoter region ( underlined , in bold ) ( fig1 ). primer b4405 consists of a 5 ′ region of sequence that is complementary to the region overlapping the start codon of endolysin from phi33 , followed by sequence that is complementary to the end of the endolysin promoter region ( underlined , in bold ; fig1 ). primer b4406 is the reverse complement of primer b4405 ( see also fig1 ). primer b4407 consists of a 5 ′ bglii restriction site ( underlined ), followed by sequence complementary to the end of the phi33 endolysin gene ( fig1 ). 2 . plasmid psmx414 ( fig1 ), comprising psmx413 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 b4408 and b4409 ( fig1 ). the resulting pcr product may then be digested with bglii and nhel , and ligated to psmx413 that has also been digested with bglii and nhel , to yield plasmid psmx414 ( fig1 ). primer b4408 consists of a 5 ′ bglii restriction site ( underlined ), followed by sequence of the lac promoter ( fig1 ). primer b4409 consists of a 5 ′ nhel 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 and the escherichia coli laczδm15 allele immediately downstream of the phoa locus of the bacterial genome 1 . plasmid psmx414 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 counter - selection , by plating onto medium containing 10 % sucrose . 3 . isolates growing on 10 % sucrose may then be screened by pcr to confirm that endolysin and laczδm15 have been introduced downstream of the p . aeruginosa phoa gene . 4 . following verification of an isolate ( pax41 ), this strain may then be used as a host for further modification of bacteriophage , where complementation of a δendolysin , laczα + genotype is required . construction of a plasmid to replace the endolysin gene of the double - tail fibre phage ( ptpx44 , ptpx45 , ptpx46 ), or similar bacteriophage , or the triple - tail fibre phage ( ptpx48 ), or similar bacteriophage , with rpsb - sasp - c and laczα 1 . plasmid psmx415 ( fig1 ), 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 b4465 and b4466 ( fig1 ). this pcr product may then be cleaned and digested with ndei and nhel . the region of phi33 sequence immediately upstream of the endolysin gene may be amplified by pcr using primers b4467 and b4468 ( fig1 ). this second pcr product may then be cleaned and digested with ndei and nhel . the two pcr product digests may then be cleaned again and ligated to psm1080 that has been digested with nhel 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 b4465 and b4468 , and by restriction digest of the purified plasmid dna with ndei , to identify plasmid psmx415 ( fig1 ). primer b4465 consists of a 5 ′ nhel restriction site ( underlined ), followed by phi33 sequence located approximately 340bp downstream of the phi33 endolysin gene ( fig1 ). primer b4466 consists of 5 ′ ndei and kpni restriction sites ( underlined ), followed by sequence of phi33 that is located immediately downstream of the endolysin gene ( fig1 ). primer b4467 consists of a 5 ′ ndei restriction site ( underlined ), followed by sequence that is complementary to sequence located immediately upstream of the phi33 endolysin gene ( fig1 ). primer b4468 consists of a 5 ′ nhel site ( underlined ), followed by phi33 sequence that is located approximately 340 bp upstream of the endolysin gene ( fig1 ). 2 . plasmid psmx416 ( fig1 ), comprising psmx415 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 b4469 and b4470 ( fig1 ). the resulting pcr product may then be digested with kpni and ncol . the rpsb promoter may be amplified by pcr from p . aeruginosa using primers b4471 and b4472 ( fig1 ). the resulting pcr product may then be digested with ncol and ndei . the two digested pcr products may then be cleaned and ligated to psmx415 that has been digested with kpni and ndei , yielding plasmid psmx416 ( fig1 ). primer b4469 comprises a 5 ′ kpni 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 ) ( underlined , in bold ; fig1 ). primer b4470 comprises a 5 ′ ncol restriction site ( underlined ), followed by sequence of the 5 ′ end of the sasp - c gene from b . megaterium strain km ( atcc 13632 ) ( fig1 ). primer b4471 comprises a 5 ′ ncol restriction site ( underlined ), followed by sequence complementary to the end of the rpsb promoter from p . aeruginosa pao1 ( fig1 ). primer b4472 comprises a 5 ′ ndei restriction site ( underlined ), followed by sequence of the beginning of the rpsb promoter from p . aeruginosa pao1 ( fig1 ). 3 . plasmid psmx417 ( fig1 ), comprising psmx416 containing laczα , may be constructed as follows . laczα may be pcr amplified using primers b4473 and b4474 ( fig1 ). the resulting pcr product may then be digested with kpni and ligated to psmx416 that has also been digested with kpni and treated with alkaline phosphatase prior to ligation , to yield psmx417 ( fig1 ). primer b4473 consists of a 5 ′ kpni restriction site ( underlined ), followed by sequence complementary to the 3 ′ end of laczα ( fig1 ). primer b4474 consists of a 5 ′ kpni restriction site ( underlined ), followed by sequence of the lac promoter driving expression of laczα ( fig1 ). genetic modification of the double - tail fibre phage ( ptpx44 , ptpx45 , ptpx46 ), or similar bacteriophage , or the triple - tail fibre phage ( ptpx48 ), or similar bacteriophage , to replace endolysin with rpsb - sasp - c and laczα 1 . plasmid psmx417 ( fig1 ) may be introduced into p . aeruginosa strain pax41 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta48 . 2 . strain pta48 may be infected in individual experiments with one of the double - tail fibre phage ( ptpx44 ( fig6 ), ptpx45 ( fig7 ), ptpx46 ( fig8 )), or similar bacteriophage , or the triple - tail fibre phage ( ptpx48 ; fig1 ), or similar bacteriophage , 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 pax41 , 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 , ptpx49 ( fig6 ), ptpx50 ( fig7 ), ptpx51 ( fig8 ), ptpx52 ( fig1 ), the isolates may be plaque purified twice more on p . aeruginosa strain pax41 , prior to further use . genetic modification to remove the laczα marker from ptpx49 , ptpx50 , ptpx51 , ptpx52 and similar derivatives of phi33 that carry rpsb - sasp - c in place of the endolysin gene 1 . plasmid psmx416 ( fig1 ) may be introduced into p . aeruginosa strain pax41 by conjugation , selecting transconjugants on the basis of tetracycline resistance ( 50 μg / ml ), yielding strain pta49 . 2 . strain pta49 may be infected in individual experiments with phage ptpx49 , or ptpx50 , or ptpx51 , or ptpx52 , 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 pax41 , onto medium containing s - gal , looking for clear 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 , ptp213 ( fig1 ; fig1 ), ptpx53 ( fig1 ; fig1 ), ptpx54 ( fig1 ; fig1 ), ptpx55 ( fig1 ; fig1 )), the isolates may be plaque purified twice more on p . aeruginosa strain pax41 , 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 (−) 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 (−) 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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