Difference between revisions of "Part:BBa K1182425"

Latest revision as of 01:30, 3 October 2013

Split β-galactosidase - α fragment

This part is an improved function of part BBa_I732005 (containing LacZ gene). We express the LacZ gene in two separate fragment. Those two fragments will still have the enzymatic activity of β-galactoside.

This is an alpha fragment of beta galactosidase split enzyme. The enzyme beta galactosidase is expressed separately into two fragment, the alpha and omega fragment. When the alpha and omega fragment meets they will exhibit an enzymatic activity similar to the full length expressed beta galactosidase enzyme.

This split reporter can be used as biosensor using ternary complexation mediated protein complementation principle where the interaction of two proteins (in this case the alpha and omega fragment) is mediated by another protein.

Characterization

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.

Stability Assay of The Split Reporter

We perform a stability assay to test the enzyme’s activity after freezing and storage in 4ᵒ C freezer for a different length of time.

Experimental setup & protocol

1. Add α and ω fragment of β-galactosidase to different eppendorf tube.

2. Store those two tubes in 4ᵒC freezer for different length of time (2 days, 7 days, 14 days, 21 days and 28 days)

3. Thaw the tube after stored in different length of time

4. Add an equal molar of both the α and ω peptide to an eppendorf tube. Incubate those tubes at room temperature on an orbital rocker for 1 hour.

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

6. The eppendorf tubes then incubated at room temperature for 3 hours

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

8. The absorbance is the analysed using 420 nm light

9. Results are expressed as percent signals obtained from freshly expressed enzyme after 3 hours of reaction

Result

Interpretation

The activity of beta-galactosidase enzyme is still above 95% after 28 days of storage. This data suggest that split reporter can be stored for a quite long time in 4ᵒC.

Sequence and Features