Design Space Exploration of the Violacein Pathway in Escherichia coli Based Transcription Translation Cell-Free System (TX-TL)
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.
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license. bioRxiv preprint first posted online Sep. 27, 2015. We thank Michael E. Lee and the Dueber Lab from UC Berkeley for providing us the yeast plasmid with the violacein pathway. We thank Dr. Nathan Dalleska and the Environmental Analysis Center for the support and assistance using LC/MS. We thank Dr. David A. Tirrell and Samuel Ho for guidance on the analysis of violacein. We thank Yutaka Hori and Clarmyra Hayes for the "E31" extract preparation. This work was supported by Caltech's Student-Faculty Program and by the Gordon and Betty Moore Foundation through Grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative.
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