Razvoj vektorja za komplementacijo prolinske avksotrofije v bakteriji Escherichia coli

Hren, Jošt (avtor), Dolinar, Marko (mentor)

Industrija se odmika od uporabe selekcijskih sistemov, ki temeljijo na odpornosti proti antibiotikom. Kljub učinkovitosti in enostavnosti ima uporaba antibiotikov v industrijski praksi več pomanjkljivosti: predstavlja dodaten strošek, obremenjuje metabolizem celice, lahko zmanjša produktivnost in prinaša tveganje za širjenje genov za odpornost v okolje ali celo na patogene mikroorganizme. Te omejitve so spodbudile razvoj alternativnih strategij za stabilno vzdrževanje plazmidov brez uporabe antibiotikov. V magistrskem delu smo pripravili avksotrofni selekcijski sistem, ki temelji na prolinski avksotrofiji. Za pripravo avksotrofnih sevov smo skušali okvariti gene, ki sodelujejo v biosintezni poti prolina: proB, proA in proC. Za izbris teh genov smo uporabili homologno rekombinacijo s sistemom lambda Red. Zato smo sestavili kasete s 350 bp dolgimi homolognimi regijami navzgor in navzdol od tarčnega prolinskega gena. Te smo z elektroporacijo vnesli v E. coli z vektorjem pKM208, ki je nosilec komponent lambda Red: Gam, ki inhibira nukleazni kompleks RecBCD in tako zaščiti linearno DNA; Exo, ki z eksonukleazno aktivnostjo razgradi 5´-konce linearne DNA in tako ustvari daljše lepljive konce; ter Beta, ki se veže na ustvarjene enoverižne konce in spodbuja prileganje homolognih zaporedij. Kot selekcijski sistem smo gen proC in operon proBA pod lastnim promotorjem in terminatorjem vstavili v vektor. S tem smo komplementirali avksotrofne seve, ki so pridobili sposobnost rasti na minimalnem gojišču. Tako smo pripravili osnovo za selekcijski sistem brez antibiotikov, ki se lahko uporablja za stabilno vzdrževanje plazmidov v E. coli. Tak pristop zmanjša tveganje za horizontalni prenos genov za odpornost proti antibiotikom ter omogoča okoljsko in regulatorno bolj sprejemljive procese. Rezultati magistrske naloge so potrdili učinkovitost avksotrofnega sistema, ki temelji na prolinski avksotrofiji. Pokazali so tudi pomembnost pozitivne selekcije pri rekombinaciji, saj nam lastnih avksotrofnih sevov ni uspelo pripraviti.

• Jezik: Slovenski jezik
• Ključne besede: avksotrofni marker, E. coli, lambda Red, prolin
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2026
• PID: 20.500.12556/RUL-180742
• COBISS.SI-ID: 273318915
• Datum objave v RUL: 16.03.2026

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Development of a vector for complementation of proline auxotrophy in Escherichia coli

Izvleček

The industry is moving away from the use of selection systems based on antibiotic resistance. Despite their efficiency and simplicity, the use of antibiotics in industrial practice has several drawbacks: they represent an additional cost, burden the cell metabolism, can reduce productivity and cause resistance gene spreading effect. These limitations have stimulated the development of alternative strategies for the stable maintenance of plasmids without the use of antibiotics. In the master's thesis, we set up an auxotrophic selection system based on proline auxotrophy. To prepare auxotrophic strains, we focused on genes involved in the proline biosynthesis pathway: proB, proA and proC. To delete these, we used homologous recombination with the lambda Red system. Therefore, we assembled cassettes with 350 bp long homologous regions upstream and downstream of the target proline genes. These were introduced into E. coli via electroporation with the pKM208 vector, which carries the lambda Red component: Gam, which inhibits the RecBCD nuclease complex and thus protects linear DNA; Exo, which degrades the 5'-ends of linear DNA with its exonuclease activity, thus creating longer sticky ends; and Beta, which binds to the created single-stranded ends and promotes the alignment of homologous sequences. As a selection system, we inserted the proC gene and the proBA operon under its native promoter and terminator into a vector. In this way, we complemented the auxotrophic strains and enabling their growth on minimal medium. Thus, we prepared the basis for an antibiotic-free selection system that can be used for stable maintenance of plasmids in E. coli. Such an approach to reducing the risk of horizontal transfer of antibiotic resistance genes and allows for better environmental and regulatory acceptability of the process. The results of the master's thesis confirmed the effectiveness of the auxotrophic system based on proline auxotrophy. They also showed the importance of positive selection in recombination since we were unable to generate our own auxotrophic strains.

• Ključne besede: auxotrophic marker, E. coli, lambda Red, proline