Određivanje količine proteina Cas3 u ovisnosti o jačini promotora, temperaturi i šaperonu HtpG u bakteriji Escherichia coli

master's thesis

• Title (english): Determination of the amount of Cas3 protein depending on the strenght of the promoter, temperature and chaperone HtpG in Escherichia coli
• Author: Lea Barbarić
• Mentor: Ivana Ivančić Baće (mentor)
• Committee member: Marko Močibob (predsjednik povjerenstva)
• Committee member: Ivana Ivančić Baće (član povjerenstva)
• Committee member: Đani Škalamera (član povjerenstva)
• Committee member: Branimir Bertoša (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2021-07-15, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

Zbog učestalih napada bakteriofaga, veliki udio bakterija razvilo je specifičan obrambeni sustav
adaptivne imunosti koji se naziva CRISPR-Cas. Sustav se sastoji od seta gena cas i lokusa
CRISPR koji se sastoji od kratkih ponavljajućih sljedova između kojih se ugrađuju razmaknice
koje potječu od strane DNA. U bakteriji Escherichia coli efikasnost zaštite bakterija od faga
sustavom CRISPR-Cas ovisi o aktivnosti proteina Cas3 koja pak ovisi o temperaturi inkubacije
i šaperonu HtpG. U ovom radu istraženo je utječe li šaperon HtpG na količinu proteina Cas3 u
ovisnosti o temperaturi i jačini promotora. Uz to, istraženo je da li promjene u konformaciji
šaperona HtpG utječu na aktivnost proteina Cas3, odnosno na efikasnost zaštite od infekcije.
Rezultati sugeriraju da šaperon HtpG utječe na funkcionalne količine proteina Cas3 u ovisnosti
o temperaturi, dok točkaste mutacije u proteinu HtpG nemaju utjecaja na interakciju HtpG s
proteinom Cas3. Jačina promotora za gen cas3 je ključna i analiza količina proteina Cas3 je
moguća samo nakon ekspresije s inducibilnog promotora.

Abstract (english)

Due to frequent bacteriophage attacks, a large group of bacteria have developed a specific
adaptive immunity defense system called CRISPR-Cas. The system consists of a set of cas
genes and CRISPR locus consisting of short repeats between which spacers derived from
foreign DNA are inserted. In Escherichia coli, the effectiveness of immunity against phages
depends on the activity of Cas3, which depends on the incubation temperature and HtpG
chaperone. In this work, the influence of HtpG chaperone on the amount of Cas3 protein as a
function of temperature and promoter strength was investigated. In addition, it was investigated
whether conformational changes of HtpG affect the activity of Cas3, ie the effectiveness of the
immunity against infections. The results suggest that chaperone HtpG affects functional
amounts of Cas3 protein as a function of temperature, whereas point mutations in HtpG protein
have no effect on the interaction of HtpG with Cas3. The strength of the promoter for the cas3
gene is critical and analysis of the amount of Cas3 is only possible from an inducible promoter.

• Keywords: CRISPR-Cas; Cas3; temperatura; fag lambda; HtpG; Escherichia coli
• Keywords (english): CRISPR-Cas; Cas3; temperature; phage lambda; HtpG; Escherichia coli
• Language: croatian
• URN:NBN: urn:nbn:hr:217:291835
• Project: Number: IP-2016-06-8861; Title: Cas3 kao kontrolna točka obrane CRISPR-Cas: razjašnjenje njegove regulacije istraživanjem stabilnosti proteina i prepisivanja u bakteriji Escherichia coli; Title: Cas3 as a key checkpoint for CRISPR-Cas immunity: Elucidating its regulation via protein stability and transcriptional control in Escherichia coli; Acronym: Cas3 status; Leader: Ivana Ivančić Baće; Jurisdiction: Croatia; Funder: HRZZ; Funding stream: IP
• Study programme: Title: Graduate university studies in chemistry; specializations in: Research programme; Course: Research programme; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2022-07-15
• Created on: 2021-09-02 12:18:34