Uvedba sistema BglBrick za združevanje genov v mlečnokislinskih bakterijah Lactococcus lactis

Ključevšek, Tim (author), Berlec, Aleš (mentor)

Mlečnokislinske bakterije so kot celične tovarne pomemben ekspresijski sistem za pridobivanje in dostavo proteinov. Ker imajo znaten biotehnološki potencial in zanimanje zanje narašča, se pojavlja potreba za razvoj novih orodij, ki bi izboljšala njihovo gensko spreminjanje. V zadnjih dveh desetletjih so razvili mnogo novih sistemov za združevanje genov, ki so olajšali in izboljšali sestavljanje genskega materiala. Standard BglBrick je ena od tovrstnih na novo razvitih metod, ki se je pojavila z optimizacijo in razvojem sistema BioBrick za združevanje genov. Temelji na ponovljivih restrikcijah in ligacijah z uporabo dveh različnih restrikcijskih endonukleaz, ki generirata kompatibilna kohezivna lepljiva konca. Ta po združitvi tvorita t. i. brazgotino v zaporedju DNA, ki je zato prej uporabljena encima ne moreta več razrezati. Sistem BglBrick za združevanje genov je bil prvotno razvit za bakterijo Escherichia coli, v okviru te magistrske naloge pa smo ga želeli prilagoditi za modelno mlečnokislinsko bakterijo Lactococcus lactis. Na osnovi plazmida pNZ8148 smo pripravili nov plazmid pNBBX, ki izkorišča restrikcijski endonukleazi BglII in BclI za tvorbo lepljivih koncev z zaporedjem GATC. Plazmid pNBBX kodira kaseto NheI-BglII-gen-BclI-XhoI, pri sestavljanju dveh kaset pa med njima nastane brazgotina TGATCT. Za preverjanje izražanja genov v plazmidu pNBBX smo uporabili tri modelne proteine: infrardeči fluorescentni protein, luciferazo NanoLucTM in afinitetno telo za receptor človeškega epidermalnega rastnega dejavnika 2. V pripravljen plazmid pNBBX smo z uporabo klasičnega kasetnega kloniranja vstavili gene za omenjene tri modelne proteine. Njihovo izražanje smo določali z merjenjem fluorescence in luminiscence ter z analizo s pretočno citometrijo. Izražanje proteinov smo ugotavljali tudi s poliakrilamidno gelsko elektroforezo, v prisotnosti natrijevega dodecilsulfata, ki sta ji sledili analizi gela s prenosom po westernu in z barvanjem z barvilom Coomassie Briliant Blue. Uspešno smo potrdili izražanje vseh treh modelnih proteinov. Sistem BglBrick smo uporabili za združitev več genov v istem plazmidu BglBrick in preverili njihovo sočasno izražanje, vendar smo bili pri tem neuspešni. Elektroferogrami, ki smo jih naredili po določevanju nukleotidnega zaporedja plazmidov so namreč vsebovali več istočasnih signalov, kar nakazuje na kontaminacijo plazmidnih vzorcev.

• Language: Slovenian
• Keywords: Lactococcus lactis, sistem BglBrick, združevanje genov
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-117441
• Publication date in RUL: 10.07.2020

Secondary language

• Language: English
• Title: Introducing BglBrick gene assembly in lactic acid bacteria Lactococcus lactis

Abstract

Lactic acid bacteria as cell factories are important hosts for production and delivery of biomolecules of interest. As they have considerable biotechnological potential and increasing interest, novel tools for improvement of their genetic manipulation are in demand. A lot of new DNA assembly methods have been developed in the last two decades to facilitate and advance the gene construction. One of the introduced assembly standards is BglBrick, which emerged from optimization of BioBrick assembly. It is a method that consists of iterative DNA digestion and ligation using two different restriction enzymes that generate compatible cohesive ends. These can be ligated, thereby generating a scar sequence in DNA that cannot be digested with either of previously used enzymes. BglBrick assembly was first introduced to Escherichia coli; however, in this study we aimed to introduce it to a model lactic acid bacterium Lactococcus lactis. We constructed a new plasmid pNBBX, on the basis of pNZ8148, that employs BglII and BclI restriction enzymes, which produce GATC sticky ends. After ligation, a TGATCT scar sequence is formed between each of the two consecutive cassettes. Altogether, our plasmids encode NheI-BglII-gene-BclI-XhoI cassettes. We applied three model proteins to test their expression in Lactococcus lactis, namely near-infrared fluorescent protein, NanoLucTM luciferase and affibody with the affinity for human epidermal growth factor receptor 2. We cloned all three model protein-encoding genes in pNBBX plasmid by using standard cassette based cloning. To determine the quantity of expressed proteins we measured fluorescence and luminescence and used flow cytometry. Expression was also determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by western blotting and staining with Coomassie Briliant Blue. We successfully confirmed the expression of all three model proteins used. We also used BglBrick standard to assemble multiple genes in the same BglBrick plasmid, and test their concomitant expression but were ultimately unsuccessful. The electropherograms after sequencing showed multiple sequence signals that were most probably caused by contaminated plasmids.

• Keywords: Lactococcus lactis, gene assembly, BglBrick system