Simpulan
Gen spmoB berhasil diklon dan diekpresikan secara berlebih di E. coli BL21 (DE3) tanpa menyebabkan toksisitas. Ekspresi ini dapat dilakukan pada suhu 27
oC dan 37 oC dengan konsentrasi IPTG 0.1 mM, 0.5 mM, dan 1.0 mM yang ditambahkan ke dalam kultur. Protein spmoB rekombinan yang dihasilkan berukuran 38.9 kDa. Aktivitas oksidasi metan bakteri rekombinan adalah sebesar 0.114 mmol/mL kultur/jam.
Saran
Perlu dilakukan penelitian untuk pemurnian protein dan pelipatan ulang secara in vitro protein rekombinan spmoB, serta optimasi kondisi kultur untuk mendapatkan aktivitas oksidasi metan protein spmoB yang lebih tinggi.
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LAMPIRAN
Lampiran 1 Pembuatan sel kompeten dengan perlakuan CaCl2 (Sambrook & Russel 2001)
Sebanyak 2 mL biakan E.coli DH5α umur 12 jam dikulturkan ke dalam 40 mL media LB. Kultur diinkubasi selama 2-3 jam pada suhu 37 °C sampai nilai Optical Density (OD600) mencapai 0.45-0.5. Selanjutnya kultur diambil sebanyak 1,5 mL dan disentrifugasi pada kecepatan 5000 rpm selama 15 menit. Pelet diresuspensi dengan 1 mL bufer transformasi. Sampel diinkubasi di dalam es selama 15 sampai 30 menit, kemudian disentrifugasi pada 5000 rpm selama 5 menit pada suhu 4 °C. Pelet diresuspensi kembali dengan 250 µL bufer transformasi dan disimpan dalam es.
Lampiran 2 Komposisi gel poliakrilamida
Bahan pemisah (mL) 12.5% gel penahan (mL) 4% gel
30% (29%:1%) akrilamida/bis 4.17 0.67
1.5 bufer Tris-HCl pH 8.8 2.5 -
0.5 M Bufer Tris-HCl pH 6.8 - 1.25
Akuades 3.33 3
10% SDS 0.10
10% amonium persulfat (APS) 0.20 0.05
Tetrametil-etilendiamin
(TEMED) 0.01 0.005
Lampiran 3 Perhitungan nilai Rf marker
Pita ke- Jarak migrasi pita (cm) Rf marker Bobot molekul (kDa)
1 0.5 0.074627 200 2 1.5 0.223881 116 3 1.8 0.268657 97 4 2.6 0.388060 66 5 4.2 0.626866 45 6 5.7 0.850746 29 Jarak CBB: 6.7 cm
Lampiran 4 Kurva standar marker
y = -1,0337x + 2,3004 R² = 0,9659 0 0,5 1 1,5 2 2,5 0 0,2 0,4 0,6 0,8 1 Lo g B M Rf Marker