Effects of Eschericia coli physiology on latent period and burst size of bacteriophage T4

Bakteriofagi so virusi, ki se brez bakterij ne morejo razmnoževati. Ravno zaradi te lastnosti so v zadnjem času, ko se število odpornih bakterij na antibiotike zvišuje, ponovno postali zanimivi, saj jih lahko uporabljamo kot zdravilo za zdravljenje bakterijskih infekcij. Študije interakcij med bakterijami in fagi so zato ključnega pomena. Pri eksperimentalnem delu smo v prvem delu s šaržnim gojenjem okarakterizirali rast bakterije Escherichia coli K-12 v kompleksnem gojišču LB. V nadaljevanju smo bakterijsko kulturo gojili v kemostatu, kjer smo lahko natančno uravnavali hitrost rasti in s tem fiziološko stanje bakterije. Pri šestih različnih hitrostih redčenja (0,24-0,98h-1) smo določili razmerje med celokupno RNA in celokupnimi proteini. Rezultati so pokazali, da se razmerje linearno povečuje s povečevanjem hitrosti redčenja. Želeli smo tudi določiti morebitne razlike v velikosti bakterijskih celic med različnimi hitrostmi redčenja, vendar je bila ločljivost svetlobnega mikroskopa premajhna. V drugem delu pa smo določili vpliv fiziološkega stanja bakterijske kulture na rastne parametre faga T4. Bakterije smo pobili s kloroformom, koncentracijo fagov pa smo določili z metodo štetja plakov. Prvi rastni parameter faga, ki smo ga določali, je bila adsorbcijska konstanta. Rezultati so pokazali, da se adsorbcijska konstanta z višanjem hitrosti redčenja zmanjšuje. Latentno periodo in pomnožitveno število fagov smo določili pri poskusu enostopenjske rasti. Latentna perioda se je od najmanjše do najvišje hitrosti redčenja skrajšala za skoraj polovico (od 43 min do 25 min). Po drugi strani pa se je pomnožitveno število fagov pri enakih pogojih zvišalo iz 21 PFU/bakterijo na 57 PFU/bakterijo, kar je skoraj trikrat več kot pri najnižji hitrosti rasti.Bacteriophages are viruses that can\u27t reproduce without the presence of bacteria. In light of the recent increase in antibiotic resistant bacteria they\u27re resurfacing in contemporary research due to their potential as an effective alternative to antibiotic treatments. To achieve this goal it is crucial to research the interactions between bacteria and phages. In the first stage of experimental work we characterised the growth of bacteria Escherichia coli K-12 in complex LB medium as a batch culture. In the next stage we cultivated the bacteria in chemostat, where we were able to precisely regulate growth rate and therefore the physiology of the bacteria. RNA/protein ratio was determined at six different dilution rates (0,24-0,98h-1). From the results it is evident that the ratio is linearly increasing with the increase of dilution rate. We also wanted to determine changes in cell size at different dilution rates but due to the low resolution of the light microscope we were unable to do so. In the second stage we determined if the growth parametres of phage T4 are effected by changes in bacterial physiology. Phage titer was determined by plaque assay method. The first phage growth parameter that we determined was the phage adsorption. Results show that with increasing dilution rate the adsorption constant is decreasing. Latent period and burst size was determined with the one-step growth experiment. Latent period dropped by almost half from the lowest to the highest dilution rate from 43 min to 25 min. On the other hand the burst size increased almost threefold from the lowest to the highest dilution rate from 21 PFU/bacterium to 57 PFU/bacterium