Tittle

: CLONNING OF MULTICOPPER OXIDASE GENE FROM Ochrobactrum sp.531 AND CHARACTERIZATION OF ITS ALKALINE LACCASE ACTIVITY TOWARDS PHENOLIC SUBSTRATES : pET23a (3666 bp)

Vector Plasmid

pET23a map Antibiotik Resistence PCR reaction : Ampicilline resistance gene (beta-lactamase)

: PCR reaction was initiated by adding 2.5 U ExTaq DNA polymerase and performed at 94oC for 5 minutes, followes by 30 cyrcle of denaturizing at 94oC for 30 sec, annealing at 54oC for 45 sec, and extention with ExTaq DNA polymerase at 72oC for 90 sec.

The PCR product (1092 bp) was then recovered using gel extraction kits, inserted into pMD18-T DNA (5:1) and incubated at 16oC for 4 hours, 5 l ligation mixture was used for transforming E.coli DH5 via Calcium Chloide Method. Positive transformants were screened using LB plates supplements with 100 g/ml Ampicillin, and further confirmed by PCR in wich two primers, 531F1 and 531R1, were used. Translation gave the clone DNA 364 amino acid with Cu II, Cu III, Cu IV sites. The primers :

Isolation DNA Recombinan Plasmid

: Centrifugation : pMD18-T

Gene of Interest Enzyme polymerase Buffer

: Multicopper Oxidase (MCO) : ExTaq DNA polymerase

: 1. Buffer A (50 mM Tris-HCl (pH 8,0)) 2. Buffer B (25 mM imidazole in 50 mM Tris-HCl (pH 8,0)) 3. Buffer C (250 mM imidazole in 50 mM Tris-HCl (pH 8,0))

Prosedure a. Isolated DNA

Novel Strain Ochrobactrum sp Isolated from soil Clone DNA fragment encoding a MCO gene

Gene of Interest (MCO) b. PCR reaction 10 ng genomic DNA extract (as template) Add 10% 10 x PCR buffer Add 0,2 mM dNTPs

Add 0,4 M primers 531F1 and 531R1 primers

DNA extract + 100 l reaction solution Solution Product (1092 bp) c. Purification 1,6 kb of PCR product Ligation mixture The cloned 1602bp DNA segment insert to pET23a vector at ecoRI and Sal I sites Take 5l Transform to E. coli DH5 via Calcium Clhoride Add gel extraction kits (to recover) Add pMD-T DNA (5:1) Mix (to inserted DNA fragments to vector pMD18-T DNA) Incubated at 16oC for 4 hours Heat 94oC for 30 sec (denaturizing) Heat 54oC for 45 sec (annealing) Heat 75oC for 90 sec (Extansion) Add 2,5 ExTaq DNA polymerase Heat 94oC for 5 minutes

Positive transformants Screen using LB plates supplements Add 100 g/ml Ampicillin

Single Colony Inoculate into 5 ml LB plates + 100 g/ml Ampicillin Incubated at 37oC overnight

Overnight culture Bacterial culture crude extract -> d. Active Staining crude extract separate the crude extract with 10% native gel do electrophoresis soaked the mini gel in 30 ml of 50 mM Tris-HCl (pH 7,5 or 8,0) for 15 minutes stained in 50 mM TrisHCl buffer (pH 8,0) containing 0,02 mM DMP + 0,2 mM CuSO4 at room temperatures for 15 minutes Incubated at 23oC Calculated density when OD600 approached to 0,6 add JPTG 0,5 mM heat 23oC for 6 hours (bacterial cell were grown) centrifuging at 6000 rpm for 15 min at 4oC add 50mM Tris-HCl (pH 8,0) centrifugation at 12,000 rpm for 15 min at 4oC Ni-affinity chromatography add buffer A (50 mM Tris-HCl (pH 8,0)) washed with buffer B (25 mM imidazole in 50 mM TrisHCl (pH 8,0)) eluted with buffer C (250 mM imidazole in 50 mM TrisHCl (pH 8,0)) 5 ml transferred into 500 ml fresh LB medium supplemented with 100 g/ml Ampicillin

crude extract separated with protein