Part:BBa_J15102:Experience

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Applications of BBa_J15102

UI Indonesia 2013 iGEM team helps characterizing this part. We have done a folding time assay 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.

User Reviews

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