Part:BBa_K1202110:Experience

We need to measure enzyme activity with observing the AI-2 production from SAH by enzyme . It’s possible to measure the autoinducer concentration by ellman’s assay. Ellmans assay needs liquid culture of enzyme expressing bacteria so we preapare liquid cultures (with antibiotics) of them. After incubating liquid cultures add 0,1 mM substrate (SAH in 10X PBS containing %1 BSA) and incubated SAH added liquid cultures at room temprature for two hour. While the bacteria incubating, enzymes producted autoinducer 2. After incubating, santrifujed and measure the supernatants. We measure autoinducer concentration, enzymes production. We use transformed bacteria as a negative control.

Ellman’s assay result:

As you see in the grafic, His-Enzyme complex and ezyme complex produced Auto Inducer-2 from the SAH. This result telling us enzymes are expressed by E.coli and succesfully produced autoinducer-2.

To observe inducible promoters response, we co incubated two different bacteria. The first bacteria culture is expressing enzyme system and the second culture is including inducible promoter.

We saw the red color at the pellet. So, it means the enzyme system is working and inducible promoter is succesfully induced from AI-2.

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