Optimizacija metode kemoporacije za vnos plazmidov v bakterijske celice : diplomska naloga

Bošnjak, Maša (author), Štrukelj, Borut (mentor)

Danes si raziskav v molekularni biologiji in genetiki ne moremo predstavljati brez tehnik rekombinantne DNA tehnologije. Ključni korak v tem procesu predstavlja vnos genskega materiala v celice. V ta namen so bile razvite številne tehnike vnosa, kljub temu pa iskanje novih, učinkovitih metod za transformacijo določene vrste bakterijskih celic predstavlja velik izziv. V diplomskem delu smo se osredotočili na optimizacijo metode kemoporacije. Kompetentne celice E. coli smo pripravili po postopku s kalcijevim kloridom. Kot možne kemoporante smo preizkusili osem spojin, za katere smo domnevali da imajo ugodne lastnosti za kemoporacijo (reverzibilno vplivajo na permeabilizacijo celične membrane). Le-te smo preizkusili v treh različnih koncentracijah, da bi dobili optimalno uspešnost transformacije (TE). Kot referenčno metodo transformacije kompetentnih celic smo uporabili metodo toplotnega šoka. Poleg optimizacije koncentracije kemoporantov smo skušali ugotoviti, kako se z velikostjo in količino plazmida, ki je na voljo, spreminja uspešnost transformacije. Metodo kemoporacije smo skušali uporabiti tudi na L. lactis. S pomočjo pretočne citometrije pa smo skušali razložiti mehanizem kemoporacije. Kemoporacija E. coli z najboljšimi kemoporanti je dajala visoke vrednosti TE, medtem ko bakterij L. lactis s to metodo nismo uspeli transformirati. Natančen mehanizem kemoporacije še vedno ostaja neznan.

• Language: Slovenian
• Keywords: kompetentne celice kemoporacija metode gelska elektroforeza biološke metode plazmidi
• Work type: Undergraduate thesis
• Typology: 2.11 - Undergraduate Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [M. Bošnjak]
• Year: 2011
• Number of pages: VII, 57 f.
• PID: 20.500.12556/RUL-121157
• UDC: 542+577.2
• COBISS.SI-ID: 2972785
• Publication date in RUL: 30.09.2020

Secondary language

• Language: English
• Title: Optimization of chemoporation for transfer of plasmids into bacterial cells

Abstract

Nowadays it is very hard to perform any research in the field of molecular biology and genetics without recombinant DNA technology. The key step of this process is the insertion of genetic material into cells. Many methods have been developed to achieve this goal. Nevertheless the discovery and development of new efficient transformation techniques remain a challenge. The main goal of this thesis was optimization of chemoporation method. Competent E. coli cells were prepared using a calcium chloride procedure. Eight different substances were tested as possible chemoporants. It was assumed that all of them had the ability to reversibly permeabilise cell membranes. Different concentrations of the substances were used in order to achieve optimal transformation efficiency (TE). The heat shock was used as reference testing method. In addition we attempted to assess the influence of plasmid size and amount on the transformation efficiency. Furthermore we tried to use the chemoporation method also for transformation of L. lactis cells. Flow cytometry was used in the attempt to explain the chemoporation mechanism. We discovered that chemoporation of E. coli with the best chemoporant substances among selected candidates gave a high value of transformation efficiency. However it was not possible to achieve transformation of L. lactis cells with the same method. Despite the attempt to explain it, the exact mechanism of chemoporation remains unclear.