Iskanje in izražanje lizinov bakteriofagov bakterij vrste Staphylococcus epidermidis in Propionibacterium acnes

magistrsko delo

Boštjan Čibej (Author), Polona Jamnik (Reviewer), Tomaž Accetto (Mentor), Tomaž Accetto (Thesis defence commission member), Matjaž Peterka (Thesis defence commission member), Mojca Narat (Thesis defence commission member), Polona Jamnik (Thesis defence commission member), Matjaž Peterka (Co-mentor)

Abstract

Lizini so bakteriofagni encimi, ki hidrolizirajo celično steno bakterij pri litičnem ciklu fagnega razmnoževanja. Namen magistrske naloge je bil vzpostaviti produkcijski sistem za heterologno izražanje lizinov, ki bi bili litični za bakterijski vrsti Propionibacterium acnes in Staphylococcus epidermidis. Ti dve vrsti sta znani kot povzročiteljici bolnišničnih okužb in nekaterih bolezenskih stanj pri človeku. V ta namen smo iz bakteriofagov, ki okužujejo ti bakterijski vrsti, izolirali lizinske gene z verižno reakcijo s polimerazo (PCR). Pri fagu E1, specifičnem za bakterije vrste P. acnes, nam ni uspelo pridobiti celotne genske sekvence lizina, zato lizinov pri tej bakteriji nismo izražali. Pri stafilokoknem fagu K (StphK) smo pridobili tri lizinske gene: 114, 154 in 156. Z rekombinantnimi tehnikami smo jih klonirali v plazmidna vektorja pQE-80L in pMAL-C5X in s transformacijo vstavili v ekspresijska seva E. coli (Top10 in Er2523). Plazmide z vključki, ki so jih vsebovale transformante, smo sekvencirali, da bi pridobili lizinske gene brez mutacij. Skupno smo pridobili trinajst lizinskih genov v obeh vektorjih. Gene z minimalnim številom mutacij smo uporabili za izražanje. Da bi čim bolj zmanjšali število mutacij v najdaljšem genu 114, smo pri PCR uporabili polimerazo Phusion, ki velja za natančnejšo od polimeraze Taq, a se to ni izkazalo za uspešno. Izražanje je bilo uspešno pri vključkih v pMAL-C5X, pri vključkih v pQE-80L pa ne. Pri pMAL-154 smo na poliakrilamidnem gelu zaznali avtoproteolizo, pri pMAL-114 pa proteolitsko razgradnjo. Lizine smo delno očistili iz bioprocesnega medija do stopnje celičnih lizatov. Test topnosti je pokazal, da se lizini nahajajo tako v topni kot netopni frakciji. Celični lizati z vsebovanimi lizini niso bili litični za stafilokokni sev SeC2, kar smo preverili s testom aktivnosti z metodo štetja kolonij na ploščah.

Keywords

mikrobna odpornost;lizini;Staphylococcus epidermidis;Propionibacterium acnes;ekspresijski sistem;Escherichia coli;

Data

Language:	Slovenian
Year of publishing:	2018
Typology:	2.09 - Master's Thesis
Organization:	UL BF - Biotechnical Faculty
Publisher:	[B. Čibej]
UDC:	602.3:578.347:604.4:615.33(043.2)
COBISS:	9048441
Views:	851
Downloads:	278
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Identification and expression of lysins from bacteriophages infecting Staphylococcus epidermidis and Propionibacterium acnes
Secondary abstract:	Lysins are hydrolytic enzymes encoded by bacteriophages that degrade bacterial cell wall during the lytic cycle of bacteriophage reproduction. The goal of this master thesis was to heterologously express lysins, that would display lytic activity against Propionibacterium acnes and Staphylococcus epidermidis. These bacteria are amongst the causative agents of nocosomial infections and different non-lethal medical conditions in humans. We used polymerase chain reaction (PCR) to isolate genes encoding for lysins from bacteriophages infecting these 2 bacteria. In the case of P. acnes, we could not obtain a full-length lysin gene from a P. acnes bacteriophage E1 so we did not express any P. acnes lysins in our work. On the other hand, we successfully isolated 3 lysins from a staphylococcal bacteriophage (StphK), namely 114, 154 and 156. We cloned the lysin genes into 2 plasmid vectors using recombinant DNA methods: pQE-80L in pMAL-C5X. Next, we transformed the vectors containing inserts into E. coli strains Top10 and Er2523. In order to obtain mutation-free lysin genes, we sequenced the inserts we isolated from transformant colonies (clones). In total, we sequenced 13 clones of lysins in 2 vectors. Clones with the minimal number of mutations were used for expression. Phusion polymerase did not provide a lower mutation rate than Taq polymerase, despite its proofreading ability. Expression of lysins was successful in pMAL-C5X but not in pQE-80L. We found that pMAL-154 exhibited autoproteolytic activity and that pMAL-114 was proteolytically degraded. Lysins were partially purified to crude form (cell lysates). Lysins were found to be present in both the soluble and insoluble fraction with the solubility test. To determine lytic activity of crude lysins, we used the plate count assay. None of the lysins exhibited any lytic activity against the staphylococcal strain SeC2.
Secondary keywords:	antimicrobial resistance;lysins;expression system;
Type (COBISS):	Master's thesis/paper
Study programme:	0
Thesis comment:	Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Pages:	XIV, 73 f., [9] f. pril.
ID:	10959118