Sinteza sintetičnega mikro-razdelka iz proteinov za izolacijo nukleoproteinskih kompleksov iz bakterije Escherichia coli.

Ivanuša, Petra (author), Butala, Matej (mentor)

Kompartmenizacija v bakterijah je pomembna, saj omogoči ločitev specifičnih procesov, ki potekajo v mikro-razdelkih in so ločeni od preostalih procesov v celici. Proteinski mikro-razdelki predstavljajo pomembno orodje v sintetični biologiji. Namen naše raziskave je bil sintetizirati proteinski mikro-rezdelek Pdu iz bakterije Citrobatcter freudii v bakteriji Escherichia coli. V ta namen smo pripravili derivat plazmida pACBSR-DL1, ki je vključeval gene operona pdu pod kontrolo promotorja pBAD, ki se aktivira ob dodatku L-arabinoze. Slednji plazmid, sicer pod kontrolo istega promotorja nosi zapis za še dva proteina, prvi nam omogoči sprostitev izbranega nukleoproteinskega kompleksa iz genoma, da bi ga lahko vključili v mikro-razdelek, drugi protein pa stabilizira DNA v kompleksu. V plazmid pACBSR-DL1 smo v več korakih klonirali nukleotidno zaporedje za proteine PduA-B-J-K-N-U, ki gradijo mikro-razdelek Pdu. Za izolacijo rekombinantnega mikro-razdelka smo uporabili gradientno centrifugiranje in s poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecilsulfata analizirali izolirane proteine.

• Language: Slovenian
• Keywords: mikro-razdelek, Gam, »Strep tag«, Pdu
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2018
• PID: 20.500.12556/RUL-103524
• Publication date in RUL: 19.09.2018

Secondary language

• Language: English
• Title: Production of synthetic protein microcompartment for the isolation of nucleoprotein complexes from bacteria Escherichia coli

Abstract

As compartmentalization of cells allows for separation of specific processes, which take place in micro-compartments and are separated from other events in the cell, it is essential for functioning of each cell. Protein micro-compartments are present in bacteria and are an important tool for synthetic biology. In this work, we sought so synthesise protein micro-compartment Pdu from Citrobatcter freudii in Escherichia coli cells. We cloned genes for the Pdu subunits PduA-B-J-K-N-U into plasmid pACBSR-DL1 under the control of pBAD promotor which is triggered by L-arabinose. Such a plasmid construct harboured also genes that enable liberation of a selected nuleoprotein complex to be packaged into a microcompartment. We applied gradient centrifugtion to isolate the L-arabinose induced microcompartment Pdu from E. coli and analysed the isolated proteins by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS PAGE).

• Keywords: microcompartment, Gam, Strep tag, Pdu