Part:BBa_K316006:Experience

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Applications of BBa_K316006

• This part can be used as an inducible reporter enzyme.GFP was added to the N-terminus of C2,3O monomer. The C2,3O enzyme is an obligate homotetramer where all 4 subunits are required for its activity. By adding GFP at the N-terminus of the each monomer, tetramerization of the enzyme is blocked, thus rendering it inactive. This modification is important as it makes the system inducible. The cleavage of the GFP by TEV protease is the input inducer signal, means that Catechol dioxygenase monomers are released so that they can form the active tetramer.

User Reviews

UNIQe7ac1a90e573bb40-partinfo-00000000-QINU

••••• Imperial College iGEM 2010

There is about a 10-fold reduction in the rate at which the protein product of GFP-XylE gene construct catalyses the reaction in comparison to the wild type XylE gene. Graph shows production of HMS (yellow product) over time after catechol addition at time 0 minutes. Different curves represent different catechol concentration added to the cell cultures Graph shows production of HMS over time after catechol addition at time 0 minutes. Different colored curves represent different substrate concentrations added at time 0min in cell cultures. GFP-XylE expression with or without TEV protease The graph presents the data acquired in an experiment to compare HMS production of cell cultures that: 1)Express XylE gene (blue) 2)Express GFP-XylE gene construct (red) 3)Express GFP-XylE construct and TEV protease (green) The results show that TEV protease cleaves the GFP-XylE fusion protein, producing active XylE protein. This can be seen by comparing the blue (XylE) and the green (GFP-XylE in presence of TEV) curve data, as they show similar rates of production of HMS colored product. The difference in the absorbance plateau value is due to slightly different initial concentrations of catechol substrate.

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