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Please use this identifier to cite or link to this item: http://arks.princeton.edu/ark:/88435/dsp01q237hs140
 Title: Comparative Production of Lasso Peptides from Multi-Precursor Plasmids Authors: Terrell, Allyse Alexandra Advisors: Link, A. James Department: Chemical and Biological Engineering Class Year: 2014 Abstract: Lasso peptides are ribosomally synthesized, post-translationally modified natural products produced by various bacteria and known for their threaded lasso shape. Their inherent stability and wide range of bioactive properties make them interesting targets for drug discovery and design. While lasso peptides were initially discovered from activity scans, genome mining is becoming a more powerful tool for the discovery of these peptides. However, many lasso peptides initially identified via genome mining have unidentified functions making it difficult to determine optimal expression conditions. As genome mining becomes a more central tool in lasso peptide research, robust expression systems capable of producing significant yields under conditions unrelated to the natural function of the peptide are needed. This work details an attempt to increase production of astexin-2 and astexin-3 from what has previously been obtained and an exploration of the regulatory system governing lasso peptide production. Plasmids were built in which the regions surrounding the precursor genes, and any regulatory elements therein, were removed and replaced with synthetic RBS’s and spacer regions. Additionally, an attempt is made to isolate “astexin-4,” a potential lasso peptide coded upstream and antisense of atxA2 in the natural gene cluster. Six dual-precursor plasmids containing permutations of the astexin-2, -3, and -4 precursors were created in addition to one tri-precursor plasmid containing three separate copies of the astexin-3 precursor all of which were expressed in E. coli. Total peptide production was found to be highly dependent on the combination of precursor genes and the positioning of precursor as primary or secondary precursor. Although astexin-4 was not observed, the presence of the astexin-4 precursor gene in the plasmid was found to retard or completely prevent lasso peptide production in a location-dependent manner. Plasmids containing the astexin-2 and astexin-3 precursors showed a bias toward astexin-3 in contrast to the natural gene cluster which produces more astexin-2. While increased production was not achieved, strides were made in uncovering the function of the astexin-4 open reading frame as well as elucidating the complexity of the regulation in the natural gene cluster. Extent: 79 pages URI: http://arks.princeton.edu/ark:/88435/dsp01q237hs140 Type of Material: Princeton University Senior Theses Language: en_US Appears in Collections: Chemical and Biological Engineering, 1931-2016

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