The impact of different genes on the CRISPR-Cas mediated defence in Escherichia coli

Sustav CRISPR-Cas je važan obrambeni mehanizam mnogih prokariota od infekcije stranom DNA, poput plazmida i faga. Sastoji se od lokusa CRISPR i 8 gena cas u neposrednoj blizini. Lokus CRISPR sastoji se od niza ponavljajućih palindromskih sekvenci između kojih se nalaze razmaknice, podrijetlom iz faga ili plazmida. Proteini Cas sudjeluju u svim fazama zaštite bakterija od napada stranom DNA koji se odvija u 3 faze. Sekvenciranjem transkriptoma bakterije Escherichia coli uzgojenih u različitim uvjetima rasta, pronađeni su zanimljivi geni kandidati koji bi mogli utjecati na aktivnost sustava CRISPR-Cas. U ovom radu je istraživano utječu li geni cspE, ibpB, oppA, symE, ygcU i yjfN na aktivnost sustava CRISPR-Cas u zaštiti bakterije E. coli. Istraživani geni su uspješno klonirani u ekspresijski vektor pBad-HisA što je potvrđeno metodom PCR, restrikcijom plazmida i sekvenciranjem. Tri gena su uspješno deletirana. Analiza dobivenih rezultata ukazuje da niti jedan od istraživanih gena nije aktivator niti represor sustava CRISPR-Cas. Geni symE i yjfN povratili su aktivnost sustava CRISPR-Cas na 30 °C u soju bez gena hns i htpG što ukazuje na njihovu indirektnu ulogu. Samo gen cspE pokazao je utjecaj na naivnu adaptaciju.The CRISPR-Cas system is an important defense mechanism of many prokaryotes by DNA infection, such as plasmids and phages. It consists of the CRISPR locus and 8 cas genes in the immediate vicinity. The CRISPR locus consists of a series of repetitive palindromic sequences sperated by spacers which are derived from phages or plasmids. Cas proteins participate in all phases of protection of bacteria from attack by foreign DNA, which takes place in 3 phases. By sequencing Escherichia coli transcripts grown under different growth conditions, interesting candidate genes were found. We hypotheised that these genes could affect the activity of the CRISPR-Cas system. In this work, the role of cspE, ibpB, oppA, symE, ygcU and yjfN genes were tested in the CRISPR-mediated protection of E. coli. The investigated genes were successfully cloned into the pBad-HisA expression vector and confirmed by PCR, restriction of plasmids and sequencing. Three genes were successfully deleted. Analysis of the obtained results showed that none of the candidate genes is an activator or repressor of the CRISPR-Cas system. The symE and yjfN genes restored activity of the CRISPR-Cas system at 30 °C in a strain lacking the hns and htpG genes, indicating their indirect role. Only the cspE gene showed an effect on naive adaptation