Difference between revisions of "Phage therapy"

1,296 bytes removed ,  10:56, 21 August 2015
no edit summary
 
(8 intermediate revisions by 4 users not shown)
Line 10: Line 10:
:'''Craig Rouskey''', MSc in Molecular Biology, Biochemistry, Microbiology and Immunology
:'''Craig Rouskey''', MSc in Molecular Biology, Biochemistry, Microbiology and Immunology
:'''Ami Knop Ullrich''', MSc in Medical Microbiology and Immunology
:'''Ami Knop Ullrich''', MSc in Medical Microbiology and Immunology
:'''Nina DiPrimio''', PhD in Pharmacology
 
:'''Louis Huang''', BSc in Chemical Engineering and Economics
:'''Marc Juul''', MSc in Biotechnology Engineering
:'''Jenny Ryan''', MA in Anthropology, MA in Communication
:'''Tess Westbrook''', BSc Biology, MBA
:'''Ianto Lin Xi''', BSc Molecular Biology


== Introduction ==
== Introduction ==
Line 103: Line 98:


== Questions and Specific Aims ==
== Questions and Specific Aims ==
:
[[Experiments]]
:'''Pilot Project Aims'''
:'''Pilot Project Aims'''
<br />
<br />
Line 112: Line 107:


:''Confirmation of N. gonorrhoeae''. Upon primary culture and propagation, N. gonorrhoeae samples will be confirmed using nucleic acid testing. A panel of polymerase chain reactions will be performed on DNA isolated from bacterial cultures. These panels will be performed to (1) identify genes responsible for 1st-, 2nd- and 3rd-generation antibiotic resistance, (2) differentiate N. gonorrhoeae from other Neisseria species. The differentiation PCR will be confirmed using CTA sugar growth testing as described by the CDC (REF). Briefly, isolates will be assessed for their ability to grow in Glucose, Maltose, Lactose and Sucrose. N. gonorrhoeae is positive for glucose metabolism, but not maltose or lactose metablism.
:''Confirmation of N. gonorrhoeae''. Upon primary culture and propagation, N. gonorrhoeae samples will be confirmed using nucleic acid testing. A panel of polymerase chain reactions will be performed on DNA isolated from bacterial cultures. These panels will be performed to (1) identify genes responsible for 1st-, 2nd- and 3rd-generation antibiotic resistance, (2) differentiate N. gonorrhoeae from other Neisseria species. The differentiation PCR will be confirmed using CTA sugar growth testing as described by the CDC (REF). Briefly, isolates will be assessed for their ability to grow in Glucose, Maltose, Lactose and Sucrose. N. gonorrhoeae is positive for glucose metabolism, but not maltose or lactose metablism.
 
<br />
:'''Isolate ARNG-derived bacteriophage from 10 patients'''.  
:'''Isolate ARNG-derived bacteriophage from 10 patients'''.  
''Bacteriophage Isolation''. Bacteriophage will be isolated from 10 clinical samples as previously described (REF). Briefly,  isolated colonies will be selected and cultured in  5 mL TPY broth (trypticase, phytone, and yeast extract) and cultued for 24 hours at 37oC in 5% CO2.
:''Bacteriophage Isolation''. Bacteriophage will be isolated from 10 clinical samples as previously described (REF). Briefly,  isolated colonies will be selected and cultured in  5 mL TPY broth (trypticase, phytone, and yeast extract) and cultued for 24 hours at 37oC in 5% CO2.
<br/>
<br />
:After culture, 1500 µL of broth will be transferred to Trypticase Soy Agar (TSA) plates in triplicate (500uL each).  One plate will be used for harvesting bacteriophage, a second plate will be used for propagating bacteriophage, and a third plate used for storing cultures.  The remaining culture (3500uL) will be centrifuged for 10min at 2500 RPM. The supernatant will be removed and filtered through a 0.22 µm syringe filter. The syringe filters removes all cellular debris resulting in a pure bacteriophage culture. Bacteriopahge cultures will then be added to a bacterial lawns using 10-fold titrations to determine titers. Positive titers will result in plaque formation.
:After culture, 1500 µL of broth will be transferred to Trypticase Soy Agar (TSA) plates in triplicate (500uL each).  One plate will be used for harvesting bacteriophage, a second plate will be used for propagating bacteriophage, and a third plate used for storing cultures.  The remaining culture (3500uL) will be centrifuged for 10min at 2500 RPM. The supernatant will be removed and filtered through a 0.22 µm syringe filter. The syringe filters removes all cellular debris resulting in a pure bacteriophage culture. Bacteriopahge cultures will then be added to a bacterial lawns using 10-fold titrations to determine titers. Positive titers will result in plaque formation.
 
<br />
:'''Characterize ARNG-derived bacteriophage from 10 patients'''.
:'''Characterize ARNG-derived bacteriophage from 10 patients'''.


Line 139: Line 134:
#Addressing previous citizen science failures and conducting research with a greater adherence to quality.
#Addressing previous citizen science failures and conducting research with a greater adherence to quality.
#Preventing Lysogeny.
#Preventing Lysogeny.
== Experiments ==
:Samples will be grown on Modified Thayer Martin agar supplemented with antibiotics. Colonies will '''(1)'''be stored in glycerol at -80oC, '''(2)'''grown in liquid culture for the assessment of bacteriophage production, '''(3)'''grown in Maltose to ensure we have cultured ''N. gonorrhoeae,'' and '''(4)'''grown in Fastidious Broth with 3rd-generation antibiotics to assess antibiotic resistance.
:
:Following growth in FB, supernatants will be analyzed for the presence of bacteriophage using SDS-PAGE electrophoresis.
:
:If bacteriophage proteins are present in the supernatants, these cultures will be re-grown in bulk and bacteriophage-containing sups will be stored at -80oC.
:
:All clinical screening data will be available to patients online through our website. If patients are positive, they will be asked to fill out a form on their results page that will detail their risk level. This data is important in the surveillance of antibiotic-resistant ''N. gonorrhoeae'' emergence. '''''All screening procedures and results are anonymous.'''''


===Protocol Documentation===
===Protocol Documentation===
http://aegia.nu/130721_prephage-protocols.txt
http://aegia.nu/folder/getitproject/130721_prephage-protocols.txt


== Laboratory Needs ==
== Laboratory Needs ==
Line 210: Line 196:
== References ==
== References ==
<references />
<references />
[[Category: Health]]
1,319

edits