We can simulate the isolation of 6 Pseudomonas phages from any site and
source (for example :from raw sewage or from other material).
The list below reports the main steps for analyzing these phages and in prospect to verify if they are utilizable for phage therapy :
1- Host growth
In this case the host is Pseudomonas aeruginosa.
Remember: bacteria and phages must be isolated from the same country . This a fundamental rule and it is an hard-and-fast rule.
2-Isolation,propagation,purification and concentration of Pseudomonas phages
3- Classification in lytic or lysogenic phages
Remember: Phages must be (at moment ) only Lytic phages. This a fundamental rule and it is an hard-and-fast rule.
4-Pseudomonas phage DNA isolation
5- Restriction Enzyme digestion
6-RAPD PCR analysis
7-SDS or 2D SDS PAGE
8- Morphology by Electron Mycroscopy
From the NCBI genome site I have chosen by random selection 6 Pseudomonas phages:
1°
-LOCUS: NC_007808,49639 bp,DNA linear; PHG: 06-MAY-2009.-ORGANISM Pseudomonas phage PA11, Viruses; dsDNA viruses, no RNA stage; unclassified dsDNA phages.
-BASES: 1 to 49639.
-HOST:Pseudomonas aeruginosa.
-AUTHORS:Kwan,T., Liu,J., Dubow,M., Gros,P. and Pelletier,J.
-SUBMITTED: Targanta Therapeutics Inc., 7170 Frederick
Banting, 2nd Floor, Ville Saint Laurent, Quebec H4S 2A1, Canada.
2°
-LOCUS: NC_011107,42961 bp,DNA linear; PHG: 17-APR-2009.-ORGANISM: Pseudomonas phage PT2 ,Viruses; dsDNA viruses, no RNA stage; Caudovirales; Podoviridae;Autographivirinae.
-BASES: 1 to 42961.
-HOST:Pseudomonas aeruginosa.
-AUTHORS: Glonti,T., Lingohr,E.J., Kropinski,A.M. and Chanishvili,N.
-SUBMITTED: George Eliava Institute of Bacteriophage,Microbiology and Virology, 3, Gotua Street, Tbilisi 380060, Georgia.
3°
-LOCUS: NC_011105,42954 bp,DNA linear; PHG: 17-APR-2009.
-ORGANISM: Pseudomonas phage PT5 ,Viruses; dsDNA viruses, no RNA stage; Caudovirales; Podoviridae;Autographivirinae.
-BASES: 1 to 42954.
-HOST:Pseudomonas aeruginosa.
-AUTHORS: Glonti,T., Lingohr,E.J., Kropinski,A.M. and Chanishvili,N.
-SUBMITTED: George Eliava Institute of Bacteriophage,Microbiology and Virology, 3, Gotua Street, Tbilisi 380060, Georgia.
4°
-LOCUS: NC_013691,74901 bp,DNA linear; PHG: 30-DEC-2009
-ORGANISM: Pseudomonas phage LUZ7,Viruses; dsDNA viruses, no RNA stage; Caudovirales; Podoviridae; N4-like viruses; unclassified N4-like viruses.
-BASES: bases 1 to 74901
-HOST:Pseudomonas aeruginosa.
-AUTHORS: Ceyssens,P.J.
-SUBMITTED: Ceyssens P.J., Biosystems, K.U.Leuven,Kasteelpark Arenberg 21, 3000 Leuven, BELGIUM
5°
-LOCUS: NC_010326 ,43548 bp,DNA linear ; PHG: 17-APR-2009
-ORGANISM: Pseudomonas phage LUZ19,Viruses; dsDNA viruses, no RNA stage; Caudovirales; Podoviridae;Autographivirinae; unclassified phiKMV-like phages
-BASES: 1 to 43548
-HOST:Pseudomonas aeruginosa.
-AUTHORS: Ceyssens,P.J.
-SUBMITTED: Ceyssens P.J., Gene Technology, K.U.Leuven,Kasteelpark Arenberg 21, Leuven, 3001, BELGIUM
6°
-LOCUS: NC_010325,45625 bp,DNA linear ; PHG: 23-JUL-2008
-ORGANISM: Pseudomonas phage LUZ24, Viruses; dsDNA viruses, no RNA stage; Caudovirales; Podoviridae.
-BASES: 1 to 45625
-HOST:Pseudomonas aeruginosa.
-AUTHORS: Ceyssens,P.J.
-SUBMITTED: Ceyssens P.J., Gene Technology, K.U.Leuven,Kasteelpark Arenberg 21, Leuven, 3001, BELGIUM
In this list are present two phages from Tiblisi ,Georgia.
I have also considered this random selection like a true phage isolation.
The points 1,2,3 and 4 are not discussed because are obvious but the point 4 is replaced by the genome sequences.
I have generated also the RAPD primers from Pseudomonas phage PT 2.
By MAUVE software I have compared these Phages:
Three of these phages are similar and it is logic and evident for PT2 and PT5 that are isolate from Georgia but among LUZ19 ,LUZ24 and LUZ7 only LUZ19 is similar to phages from Georgia. Is this phage isolates from Belgium?
The answer is been found the day after the date of this post in this good document and my random selection was pure coincidence:
"A collection of 28 newly isolated P. aeruginosa phages was established, sampled in 14 different countries traversing four continents from both natural environments and hospital sewage. Fifteen of these phages were selected based on their ability to form clear plaques, a hallmark feature of virulent phages. All displayed unique DNA and protein profiles and clustered morphologically into six groups within the three major families of the Caudovirales. Extensive host range studies on a library
The answer is been found the day after the date of this post in this good document and my random selection was pure coincidence:
"A collection of 28 newly isolated P. aeruginosa phages was established, sampled in 14 different countries traversing four continents from both natural environments and hospital sewage. Fifteen of these phages were selected based on their ability to form clear plaques, a hallmark feature of virulent phages. All displayed unique DNA and protein profiles and clustered morphologically into six groups within the three major families of the Caudovirales. Extensive host range studies on a library
of 122 AFLP-genotyped clinical P. aeruginosa strains showed that the phages lysed 87% of the strains.
Infection analysis of outer membrane mutants identified ten phages as type IV pili-dependent. More detailed information about their molecular relatedness was gathered by de novo peptide sequencing of major virion proteins using MALDI-TOF/TOF technology. Applying this technique for the first time to viruses, seven groups of closely related phages were identified without the need of prior knowledge of genome content and/or electron microscopic imaging. Phages from five different groups were selected for further genomic and proteomic characterization"
Restriction Enzyme digestion
I have utilised SphI enzyme:
RAPD PCR analysis
inverted repeat n: 9
65 cctgggagcttccggctt 83
1766 cttgggagccttagcctt 1748
>Tm=56,9°C Tm10=32,1°C CG%=61,1 MW=5482,1 D 18bp: PCR efficiency (quality)=90
5'-cytgggagcytymgsctt
inverted repeat n: 10
76 ccggcttcgccgggt 91
9334 ccggcttcatcgcgt 9319
>Tm=57,6°C Tm10=38,0°C CG%=73,3 MW=4540,5 D 15bp: PCR efficiency (quality)=91
5'-ccggcttcrycgsgt
inverted repeat n: 17
130 cctggcgggagtgtcaa 147
509 cctagagggagtggcca 492
>Tm=56,1°C Tm10=33,9°C CG%=64,7 MW=5255,9 D 17bp: PCR efficiency (quality)=90
5'-cctrgmgggagtgkcma
inverted repeat n: 108
1129 gacacggtgcagggc 1144
4973 gccacggtggaaggc 4958
>Tm=56,3°C Tm10=39,4°C CG%=73,3 MW=4643,1 D 15bp: PCR efficiency (quality)=87
5'-gmcacggtgsarggc
inverted repeat n: 322
5381 gtcggcagcccaggc 5396
14983 atcggcagttccggt 14968
>Tm=56,0°C Tm10=37,8°C CG%=70,0 MW=4581,5 D 15bp: PCR efficiency (quality)=90
5'-rtcggcagyycmggy
inverted repeat n: 337
5673 cacgtcgctgggagat 5689
9935 cgcggcgctgggacat 9919
>Tm=57,4°C Tm10=33,8°C CG%=68,8 MW=4922,7 D 16bp: PCR efficiency (quality)=92
5'-crcgkcgctgggasat
9977 ccggcgctgatggctg 39993
42685 ctggcgctggtagcca 42669
>Tm=58,0°C Tm10=32,1°C CG%=71,9 MW=4906,2 D 16bp: PCR efficiency (quality)=90
5'-cyggcgctgrtrgcyr
37525 gacgccaacggcgat 37540
38109 gacgccaccgtccac 38094
>Tm=55,4°C Tm10=36,5°C CG%=70,0 MW=4535,0 D 15bp: PCR efficiency (quality)=85
5'-gacgccamcgkcsay
5024 cgttctcggcctgaacct 35042
36632 cgttctcgccatagagct 36614
>Tm=55,3°C Tm10=31,1°C CG%=58,3 MW=5438,6 D 18bp: PCR efficiency (quality)=97
5'-cgttctcgscmtrrasct
21728 cgcggatggcctgctg 21744
26225 cgcggattgcttgtag 26209
>Tm=55,3°C Tm10=33,3°C CG%=65,6 MW=4921,2 D 16bp: PCR efficiency (quality)=89
5'-cgcggatkgcytgywg
19627 cgacatcgccaaggc 19642
25117 ccgcatcgcccaggt 25102
Only three phages ( PT2,PT5 and LUZ19) have given PCR products.
2D SDS PAGE
Phage Coktails
