Part:BBa_K1688009
dTomato, ModLac and Dioxygenase (inc RBS, NahR/Psal promoter system) Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 786
Illegal BglII site found at 3994 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 77
Illegal NgoMIV site found at 618
Illegal NgoMIV site found at 2277 - 1000COMPATIBLE WITH RFC[1000]
BBa_J61051, nahR with nahR and sal promoters (Pr and Psal), codes for the inducible repressor NahR, which suppresses Psal in the absence of salicylate. This biobrick makes it possible to regulate the expression of the red fluorescent protein, dTomato (BBa_K1688004), the modified laccase ModLac (BBa_K1688006), and a dioxygenase.
The dTomato protein is a fluorescent protein dimer, created by direct evolution of the wild-type DsRed, from Discosoma sp. (Shaner et al, - Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein, 2004). The dTomato protein is a fluorescent dimer protein that emits orange-red light when it is excited by green-yellow light. It is preferable to use – especially in self-made fluorometry tests – because the excitation wavelengths and the emission wavelengths don't overlap as much as in other fluorescent proteins. The dTomato excitation peak is at 554 nm and 50% of it is at 510 nm. Also, its emission peak is at 581 nm and its 50% emission at 629 nm.
Laccases (originally from Chinese lacquer tree sap) are multicopper oxidases, that are employed in various industries, where they take part in beer maturation, textile dyeing, and enzymatic biofuel cells. Due to their broad specificity and ability to oxidize aromatic compounds, their application in bioremediation is a topic under investigation. The laccase we chose is a modified laccase, CueO, a laccase from E. coli.
The enzymatic activities of the laccases was measured using ABTS. ABTS is a commonly used substrate when evaluating reaction kinetics of specific enzymes. Due to its reduction potential, it acts as an effective electron donor. Since we are working with laccases, which are multi copper oxidases, which oxidize substrates, ABTS is a suitable substrate. ABTS will donate electron to reduce molecular oxygen. The oxidized ABTS has a different absorption spectrum and the reaction can thus be observed in a spectrophotometer.
Dioxygenase is a type of enzyme that uses dioxygen to catalyze the oxidation of hydrocarbons. This type of enzyme is used to catalyze cleavage of the aromatic rings in our degradation pathway. We chose to use a catechol dioxygenase which is an important part of bacterial degradation of aromatic compounds. There are two main types of catechol dioxygenase; intradiol and extradiol dioxygenase. The end product in the intradiol degradation pathway is a carboxylic acid which is unharmful to the cell and can be used in the bacteria's metabolism. The end product in the extradiol degradation pathway is an aldehyde which is much more harmful to the cell than a carboxylic acid. Therefore an intradiol dioxygenase; catechol 1,2 dioxygenase, was chosen. (Bugg, 1 September 2003) This enzyme had been used by a previous iGEM team; Hong Kong 2013 (http://2013.igem.org/Team:Hong_Kong_CUHK/backgroundPAH) but had not been characterized (BBa_K1092003) https://parts.igem.org/Part:BBa_K1092003. This specific isoform of dioxygenase is suitable for extracellular export, since most known dioxygenases are dimers, stabilized by noncovalent interactions, and the current catechol 1,2 dioxygenase is a monomer. Inferring from the crystal structure of the enzyme, both its C and N termini do not participate in critical interactions within the enzyme, which makes them suitable sites for export tag fusion.
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