Difference between revisions of "Part:BBa K1182425:Experience"

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Applications of BBa_K1182425

Activity Assay of The Split Reporter

To test the function of our split reporter, we collect the data of enzyme activity over time. We exploit the enzyme’s ability to convert ortho-Nitrophenyl-β-galactoside (ONPG), a colorless substance, into ortho-nitrophenol, a visible yellow substance. We then use a spectrophotometer to quantisize the absorbance of the solution.

Experimental setup & protocol

1. The α and ω fragment of β-galactosidase was cloned into pQE-80L and pQE-81L, respectively

2. The α and ω fragment was then expressed (in TOP10 E.coli) and purified using His tagged protein purification method

3. Add an equal molar of both the α and ω peptide to eppendorf tube #1 - #4; α fragment to eppendorf tube #5 - #8; ω fragment to eppendorf tube #9 - #12; and diluted full length β-galactosidase to tube #13 - #16. Incubate those tubes at room temperature on an orbital rocker for 1 hour.

4. At time zero, 20µL of ONPG (4mg/mL) was added into each tube

5. The eppendorf tubes then incubated at room temperature for different length of time (30 min, 90 min, 3 hours, and 19 hours).

6. The reaction was then terminated by adding 50µL 1M Na2CO3

7. The absorbance is the analysed using 420 nm light

Result

Interpretation

The full length β-galactosidase reaction mix works as a positive control while both the α-only and ω-only reaction mix works as a negative control.

The split reporter have the activity of full length β-galactosidase enzyme, while none of the α-only nor the ω-only have the enzymatic activity. This data suggests that the peptide complementation needs to occur in order to generate enzymatic activity. Previous study shows that the peptide needs many minutes to form the tetrameric structure which have the enzymatic activity. That’s why we incubate them for 1 hour after mixing the α and ω fragment.

The data also shows that the split reporter needs longer timer to digest the same amount of ONPG compared to full length β-galactosidase. This data suggest that our split reporter works as expected.

User Reviews

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