KLONING DAN EKSPRESI GEN ACETYL-COA ACETYLTRANSFERASE (phaA) PADA ESCHERICHIA COLI BL21(DE3)

SUJATA SARI TETEKI, M.Saifur Rohman, M.Si.M.Eng., Ph.D.; Prof. Ir. Irfan Dwidya Prijambada, M. Eng., Ph.D.

2021 | Skripsi | S1 MIKROBIOLOGI PERTANIAN

Polyhydroxyalkanoate (PHA) merupakan salah satu bahan sangat menjanjikan untuk plastik biodegradable. Salah satu family PHA yaitu Poli-β-hdroksibutirat (PHB) disintesis melibatkan tiga enzim utama yaitu acetyl-CoA acetyltransferase, acetyl CoA reductase dan phaC sintase. Acetyl-CoA acetyltransferase mengkatalisis dua molekul asetil-CoA menjadi asetoasetil-CoA. Asetoasetil CoA merupakan salah satu precursor penting untuk proses biosintesis polihidroksialkanoat. Tujuan penelitian ini yaitu kloning dan ekspresi open reading frame gen phaA dari Priestia megaterium PSA 14 serta mendapatkan klon dan ekspresi open reading frame gen phaA yang mengkode acetyl-CoA acetyltransferase pada bakteri E.coli BL21 (DE3). Amplifikasi ORF phaA dilakukan dengan PCR kemudian dilakukan kloning ke dalam plasmid pET28a(+) dengan menggunakan enzim restriksi NcoI dan EcoRI. Proses penempelan antara ORF gen phaA dan plasmid pET28a(+) yang terpotong menggunakan T4 ligase. Rekombinan protein dianalisis menggunakan 12% SDS PAGE. Dari penelitian ini, orf phaA berhasil diamplifikasi dan dikloning kedalam sistem pET. Rekombinan protein Orf phaA berhasil diekspresikan dan dan diproduksi dengan berat molekul ±43 kDa.

Polyhydroxyalkanoate (PHA) is one of the most promising materials for biodegradble platics. One of the PHAs families is Poly-β-hydroxybutyrate (PHB) which is synthesized by involving three main enzymes, i.e. acetyl-CoA acetyltransferase, acetyl CoA reductase and phaC synthase. acetyl-CoA acetyltransferase catalyzes the formation of acetoacetyl-CoA from two molecules of acetyl-CoA. Acetoacetyl CoA is one of important precursor for polyhydroxyalkanoate biosynthesis process. This study was aimed to clone and overexpress an open reading frame of the phaA gene (acetyl-CoA acetyltransferase encoding gene) from Priestia megaterium PSA14. The ORF of phaA gene was amplified by PCR then the PCR product was cloned into pET28a(+) following NcoI and EcoRI double digestion. The ligation process of the ORF of phaA and the plasmid was carried out by T4 DNA ligase. The ligation product was then used to transform E. coli BL21(DE3). The recombinant protein was examined by 12% SDS-PAGE. From this work, the ORF of phaA was sucessfully amplified and cloned into pET system. The ORF of phaA was sucessfully overexpressed and produce the recombinant protein with molecular weight of ±43 kDa.

Kata Kunci : E.coli BL21 (DE3), ORF phaA, Priestia megaterium, and acetyl CoA acetyl-transferase.