Hitro določanje koncentracije infektivnih bakteriofagov lambda

Umek, Barbara (author), Podgornik, Aleš (mentor)

Bakteriofag lambda je eden najbolj preučenih in uporabljenih virusov v mikrobni in molekularni genetiki. Koncentracijo lambde v vzorcu najpogosteje določamo z metodo plakov in čeprav je metoda delovno in časovno zahtevna, je možno le na tak način določiti koncentracijo infektivnih bakteriofagov. Znano je, da receptorski protein LamB bakterije S. sonnei sproži in vitro ejekcijo DNA bakteriofaga lambda, količina sproščene DNA pa sovpada s koncentracijo infektivnih bakteriofagov. Mehanizem in vitro ejekcije smo želeli uporabiti za izbrizganje DNA iz virusa ter postaviti metodo za hitro določanje koncentracije infektivnih bakteriofagov lambda, kjer spektrofotometrična analiza izločene DNA omogoča oceno števila bakteriofagov v izhodiščnem vzorcu. V ta namen smo potrebovali receptor LamB (maltoporin), ki smo ga izolirali iz bakterije E. coli LE392, saj spada v prvo biološko varnostno stopnjo in je senzitivnen organizem za bakteriofag lambda. Za test in vitro ejekcije smo uspešno pripravili ekstrakt membranskih proteinov, raztopljen v detergentu Triton X-100. Eksperimentalni podatki so pokazali, da receptor LamB ne sproži in vitro ejekcije DNA bakteriofaga, kar smo spremljali z metodo plakov. Ker je kombinacija detergentov in organskih topil v mešanici povzročila razslojevanje faz, smo se med faznimi stanji orientirali s trikotnim faznim diagramom ter spremenili razmerje reaktantov, tako da je ejekcija potekla v enofaznem območju. Rezultati eksperimenta ejekcije se niso spremenili, zato smo na tej točki zaključili z eksperimentalnim delom. Vzrok za neuspešno inaktivacijo virusa posredovano z LamB je lahko majhna a nezanemarljiva razlika med aminokislinskima zaporedjema receptorja LamB bakterij E. coli ter S. Sonnei, ki znaša 2 %.

• Language: Slovenian
• Keywords: biotehnologija, bakteriofagi, membranski proteini, izolacija proteinov, fazni diagrami
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [B. Umek]
• Year: 2017
• PID: 20.500.12556/RUL-99155
• UDC: 602.3:578.347(043.2)
• COBISS.SI-ID: 8894585
• Publication date in RUL: 24.12.2017

Secondary language

• Language: English
• Title: Fast detection and determination of lambda bacteriophage

Abstract

Lambda phage is intensively used as model organism and enabled development of many methods in microbial and molecular genetics. Concentration of lambda in sample is usually determined by plaque forming units, despite method is labor intensive, time consuming and rather inaccurate. Still, PFU is the only method for routine determination of infectious bacteriophages. It is known, that receptor protein LamB (maltoporine) from S. sonnei strain can cause in vitro ejection of lambda phage DNA, and the amount of released DNA correlates with concentration of infective phages. It was our goal to transfer this phenomena into method, that would allow detection and determination of infective lambda bacteriophage concentration. First step was cultivation of E. coli LE392 strain that requires only biosafety level 1, being sensitive to lambda phage, from which we tried to isolate receptor LamB. For the in vitro ejection test we successfully prepared protein extract from outer cell membranes. Protein LamB was dissolved in detergent triton X-100. Experimental data revealed that receptor LamB was not capable of triggering in vitro ejection of bacteriophage, which we observed by plaque forming unit test. Possible reason was also the fact that combination of organic solvents and detergents used for solubilization of receptor separated into two phases. We used triangular phase diagram to properly adjust concentration and obtain solution consisting of a single phase. Despite this change, ejection did not occur. Possible reason for unsuccessful results might be in difference in aminoacid sequence among E. coli and S. sonnei strain (although being only 2 %), which could result in loss of affinity among LamB and phage.

• Keywords: biotechnology, bacteriophages, membrane proteins, protein isolation, phase diagrams