Team:Heidelberg/Templates/Indigoidine week23 overview

From 2013.igem.org

Domain Shuffling – Indigoidine Synthetase Conversions

Besides the delH4 T-domain only the plu2642 T-domain resulted in a functional indigoidine synthetase when introduced to indC. We used our NRPS-Designer to predict the domain sequence of plu2642 and the selectivity of its A-domain and found that it is a single-module NRPS with the domain sequence A, T, TE. Its A-domain exhibits glutamine specificity, analogous to our indigoidine synthetase. Also the basic domain sequence is similar, whereas the internal oxidation domain of indC is missing in plu2642.
Another NRPS module which shows glutamine specificity is tycC2 from the Tyrocidine pathway (further described in the <a href="https://2013.igem.org/Team:Heidelberg/Project/Tyrocidine">Peptide Synthesis Project Page</a>).
To take the domain shuffling experiment even one step further, we assembled every possible domain combination involving the indC oxidation domain and an A-, T- and TE-domain from indC. plu2642 or tycC2 and transform a given, naturally occuring NRPS module into a functional indigoidine synthetase.

Engineered indC – Quantitative Assay

This week we also repeated the spectral measurement of our engineered indC variants cotransformed with single PPTases to see which combination of PPTase and T-domain is most efficient in producing indigoidine.