Ekspresi Gen Penyandi Bst DNA Polimerase di Escherchia coli BL21 (DE3)

NIKMAH NURFIKRIA, Prof. Ir. Irfan Dwidya Prijambada, M.Eng., Ph. D.; Ir. Donny Widianto, Ph.D.

2022 | Tesis | MAGISTER BIOTEKNOLOGI

Bst DNA polimerase merupakan enzim yang digunakan untuk deteksi molekuler menggunakan LAMP (loop mediated isothermal amplification) yang produksinya menggunakan Escherichia coli BL21 (DE3) dan jumlah enzim yang dihasilkan tergantung pada ekspresi gen dan vektor ekspresi yang sesuai. Penelitian ini bertujuan untuk mengetahui ekspresi gen penyandi Bst DNA polimerase large fragment (Bstpol LF) dari konstruk vektor pGEX_3X. E. coli BL21 (DE3) diperoleh dari Laboratorium Rekayasa Genetika Universitas Gadjah Mada dan E. coli TOP10 dengan gen penyandi Bstpol LF dalam vektor ekspresi pGEX_3X diperoleh dari Dushanth Seevaratnam, Open Bioeconomy Lab. Plasmid pGEX_3X Bstpol LF dalam E. coli TOP10 diisolasi dan diintroduksi ke E. coli BL21 (DE3). Sel-sel transforman yang membawa plasmid pGEX_3X Bstpol LF dikonfirmasi dengan mengisolasi plasmid dan memotongnya menggunakan enzim restriksi BamH1 serta mengamplifikasi gen penyandi Bstpol LF menggunakan Polymerase Chain Reaction (PCR). Ekspresi gen dilakukan dengan menumbuhkan sel-sel transforman yang membawa plasmid pGEX_3X Bstpol LF di media Luria Bertani (LB) cair dan promotornya diinduksi dengan isopropyl ���²-D-1- thiogalactopyranoside (IPTG). Seluruh protein E. coli BL21 (DE3) diisolasi dan dianalisis menggunakan Sodium dodecyl sulphate�¢ï¿½ï¿½polyacrylamide gel electrophoresis (SDS-PAGE). Hasil konfirmasi menggunakan analisis restriksi dan amplifikasi menunjukkan adanya pita DNA berukuran diatas 6000 bp. Koloni transforman berhasil terbentuk namun, profil protein E. coli BL21 (DE3) dari hasil elektroforesis SDS-PAGE menunjukkan bahwa tidak terlihat adanya perbedaan profil protein yang dihasilkan antara yang diinduksi IPTG dengan yang tidak diinduksi. Hal tersebut menunjukkan bahwa plasmid pGEX_3X Bstpol LF dapat diintroduksi di E. coli BL21 (DE3) dan diperbanyak namun, gen penyandi Bstpol LF belum tampak jelas terekspresi atau tidak terekspresi di E. coli BL21 (DE3).

Bst DNA polymerase is an enzyme used for molecular detection using LAMP (loop mediated isothermal amplification) which is produced by Escherichia coli BL21 (DE3) and depends on gene expression and expression vector. In this study gene Bst DNA polymerase large fragment (Bstpol LF) from vector pGEX_3X was expressed in E. coli BL21 (DE3). E. coli BL21 (DE3) was obtained from Genetic Engineering Laboratory of Gadjah Mada University and E. coli TOP10 with Bstpol LF gene in expression vector pGEX_3X was obtained from Dushanth Seevaratnam, Open Bioeconomy Lab. E. coli BL21 (DE3) was transformed with isolated recombinant plasmid pGEX_3X Bstpol LF from E. coli TOP10. Transformant cells containing plasmid pGEX_3X Bstpol LF were isolated and confirmed by digestion using restriction enzyme BamH1 and amplification gene Bstpol LF with Polymerase Chain Reaction (PCR) method then, was expressed by induction with isopropyl -D-1-thiogalactopyranoside (IPTG). E. coli BL21 (DE3) resultant proteins were analyzed using Sodium dodecyl sulphate�¢ï¿½ï¿½polyacrylamide gel electrophoresis (SDS-PAGE). The results using restriction and amplification analysis showed the presence of DNA bands above 6000 bp. Transformant colonies were successfully formed, however, the protein profile of E. coli BL21 (DE3) from SDS-PAGE electrophoresis analysis showed that there was no difference in the protein profiles produced between IPTG-induced and non-induced. This indicated that the plasmid pGEX_3X Bstpol LF could be introduced in E. coli BL21 (DE3) and propagated, however, the Bstpol LF gene was not clearly expressed or not expressed in E. coli BL21 (DE3).

Kata Kunci : E. coli BL21 (DE3), Bst DNA polimerase rekombinan, ekspresi gen.