Activity Assay

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-Beta-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 alpha and omega fragment of Beta-galactosidase was cloned into pQE-80L and pQE-81L, respectively

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

3. Add an equal molar of both the alpha and omega peptide to eppendorf tube #1 - #4; alpha fragment to eppendorf tube #5 - #8; omega fragment to eppendorf tube #9 - #12; and diluted full length Beta-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 Beta-galactosidase reaction mix works as a positive control while both the alpha-only and omega-only reaction mix works as a negative control.

The split reporter have the activity of full length Beta-galactosidase enzyme, while none of the alpha-only nor the omega-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 alpha and omega fragment.

The data also shows that the split reporter needs longer timer to digest the same amount of ONPG compared to full length Beta-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 alpha and omega fragment of Beta-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 alpha and omega 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.

Characterisation of Other Parts

Beside characterize our own part, we also characterize another old part and adding it's application. Part #BBa_J15102 and #BBa_K891003 are complementary parts that can be used to create ternary complexation mediated protein complementation based biosensor. This kind of biosensor detects a single mediator body instead of two complementing parts.

Folding Time Assay

In order to create the biosensor, first we have to test the time needed for the complementing parts to form a tetramer.

Experiment Setup and Protocol

1. Add diluted full length beta-galactosidase enzyme to eppendorf tube #1, alpha fragment to eppendorf tube #2, omega fragment to eppendorf tube #3, and mix of equimolar alpha and omega fragment to eppendorf tube #4.

2. Incubate those tubes at room temperature on an orbital rocker for 1 hour.

3. Add 20µL ONPG substrate to each tube

4. Incubate those tubes at room temperature on an orbital locker for 30 min

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

6. The absorbance is the analysed using 420 nm light

Result

Interpretation

In the first 30 minutes of reaction, full length beta galactosidase shows higher activity; split reporter shows lower activity; alpha-only and omega-only fragment shows almost no activity. This allows us to differ full length beta-galactosidase activity from split reporter activity.

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