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Design Space Exploration of the Violacein Pathway in Escherichia coli Based Transcription Translation Cell-Free System (TX-TL)

By Phuc H.B. Nguyen, Yong Y Wu, Shaobin Guo, Richard M. Murray

Posted 27 Sep 2015
bioRxiv DOI: 10.1101/027656

In this study, an Escherichia coli (E. coli) based transcription translation cell-free system (TX-TL) was employed to sample various enzyme expression levels of the violacein pathway. The pathway was successfully reconstructed in TX-TL. Its variation produced different metabolites as evident from the extracts assorted colors. Analysis of the violacein product via UV-Vis absorption and liquid chromatography-mass spectrometry (LC-MS) detected 68 nanograms of violacein per 10 microliters reaction volume. Significant buildup of prodeoxyviolacein intermediate was also detected in the equimolar TX-TL reaction. Finally, design space exploration experiments suggested an improvement in violacein production at high VioC and VioD DNA concentrations.

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