Eksperimenti za pripravo novega vektorja za kloniranje PCR-produktov z uporabo sistema toksin-antitoksin IPF_1065/1067 cianobakterije Microcystis aeruginosa PCC 7806SL

Krajnik, Bor (author), Dolinar, Marko (mentor)

Sistemi toksin-antitoksin so genetski elementi, ki so zapisani na prokariotskih plazmidih in kromosomih. Sestavljeni so iz toksina, ki lahko s svojim delovanjem povzroči propad celice, in antitoksina, ki prepreči delovanje toksina. V bakterijski fiziologiji igrajo pomembno vlogo pri obrambi pred fagnimi okužbami, tvorbi biofilmov in dedovanju plazmidov. Z uporabo toksinov iz sistemov TA v vektorjih s pozitivno selekcijo lahko po transformaciji bakterij z ligacijskimi produkti preprečimo rast celic, ki so sprejele vektor brez vključka. Če z vstavljanjem vključka uspešno prekinemo ali zamaknemo odprti bralni okvir toksina, bakterijska celica preživi, v nasprotnem primeru pa se toksin nemoteno izraža, kar vodi v propad celice. V raziskovalni skupini, v sklopu katere je bilo opravljeno to diplomsko delo, za molekulsko kloniranje PCR-produktov uporabljamo komercialni komplet reagentov. Komplet, ki vsebuje klonirni vektor s pozitivno selekcijo za uporabo v E. coli, predstavlja precejšen strošek, saj se cena za posamezno reakcijo giblje okoli 10 evrov. Da bi ta strošek zmanjšali, smo se odločili razviti nov vektor za kloniranje PCR-produktov, ki bi ga lahko proizvajali v laboratoriju. Na genomu cianobakterije Microcystis aeruginosa PCC 7806SL je kodiranih več sistemov toksin-antitoksin. Med njimi je edini okarakterizirani sistem tipa II iz naddružine RelE/ParE, ki je sestavljen iz toksina IPF_1065 in antitoksina IPF_1067. Za antitoksin je bilo predhodno dokazano močno izražanje v topni obliki v E. coli. Dokazana je bila tudi toksičnost IPF_1065 za E. coli in zmožnost IPF_1067, da nevtralizira delovanje toksina. Za pripravo novega vektorja smo poskusili zapis za toksin IPF_1065 vstaviti v sinteznobiološka vektorja pSB1C3-BBa_K608006 in pSB1C3-BBa_K608007, ki omogočata konstitutivno izražanje toksina. Da bi preprečili delovanje toksina na celice med molekulskim kloniranjem, smo v celice vstavili tudi plazmid pET-28b(+) – ipf_1067, s katerega se je inducibilno izražal antitoksin IPF_1067. Kljub temu na selekcijskem gojišču ni zrasla nobena transformanta, ki bi vsebovala katerega od novih vektorjev z zapisom za toksin. Eksperiment bi bilo smiselno ponoviti z uporabo drugačnih promotorjev za toksin in antitoksin. Lahko bi uporabili tudi toksin iz drugega sistema TA. Kloniranje bi lahko v tem primeru izvedli v bakterijskem sevu, odpornem na delovanje toksina.

• Language: Slovenian
• Keywords: sistem toksin-antitoksin, vektorji s pozitivno selekcijo, molekulsko kloniranje
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-148946
• COBISS.SI-ID: 164625667
• Publication date in RUL: 01.09.2023

Secondary language

• Language: English
• Title: Experiments for the preparation of a vector for cloning of PCR-products based on the toxin-antitoxin system IPF_1065/1067 of the cyanobacterium Microcystis aeruginosa PCC 7806SL

Abstract

Toxin-antitoxins systems are genetic elements encoded on prokaryotic plasmids and chromosomes. They are composed of a toxin, which can cause cell death, and an antitoxin that counteracts the actions of the toxin. In bacterial physiology TA systems play an important role in the defense against phage infections, biofilm formation and plasmid inheritance. Usage of toxins from TA systems in positive selection vectors allows for selective growth of desired transformants after transformation of bacterial cells with ligation products. If the open reading frame of the toxin is interrupted or shifted by an insert, the transformed cells can survive, while in the opposite case the toxin is expressed, resulting in cell death. In the research group in which this work was conducted, a commercially available kit is used for cloning PCR-products. This kit contains a specific cloning vector for use in E. coli, but its usage is quite expensive as a single reaction costs about 10 euros. To reduce costs, we decided to develop a new positive selection vector for cloning PCR-products, which can be produced in the laboratory. The genome of the cyanobacterium Microcystis aeruginosa PCC 7806SL harbors many toxin-antitoxin systems. Among them is the system IPF_1065/1067 from the RelE/ParE superfamily of type II systems, which is the only one that has been previously experimentally characterized. It is composed of the IPF_1065 toxin and IPF_1067 antitoxin. Strong expression in E. coli has previously been shown for the antitoxin. Also shown, was the toxicity of IPF_1065 to E. coli and the ability of IPF_1067 to neutralize the effects of the toxin. We attempted to insert the toxin from this system into the synthetic biology vectors pSB1C3-BBa_K608006 and pSB1C3-BBa_K608007, which would enable constitutive expression of the toxin. To counteract the unwanted effects of the toxin during assembly of the positive selection vector, we simultaneously transformed the bacteria with the vector pET-28b(+) – ipf_1067, from which expression of the antitoxin IPF_1067 could be induced. However, no transformants containing the new vectors with the toxin sequence grew on the selection medium. We suggest that further experiments focus on different promoters for the expression of the toxin and antitoxin. Other toxins from different TA modules could be tried out. In this case, cloning of the toxin could be carried out in a bacterial strain that is resistant to the effects of the toxin.

• Keywords: toxin-antitoxin system, positive selection vectors, molecular cloning