Browsing by Subject "Plasmid annotation"
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Item Engineered plasmids : uncovering lost histories and improving annotation(2023-04-13) McGuffie, Matthew James; Barrick, Jeffrey E.; Wilke, Claus O; Marcotte, Edward M; Davies, Bryan WEngineered plasmids are a fundamental tool in many biological disciplines. They are the primary workhorses for molecular cloning, protein expression, genetic engineering, and more. Their utility stems from the fact that they are relatively easy to build using a standard set of well-understood DNA sequences. However, since engineered plasmids are created by scientists, they may contain artifacts from their construction histories that affect their functions in unexpected ways. Furthermore, engineered plasmids tend to receive less attention regarding the evolutionary pressures they face, compared to natural DNA sequences. In particular, the backbones of these plasmids, containing origins of replication and selection markers, are typically assumed to be immutable or at least tolerant of mutations. Nonetheless, engineered plasmids are subject to the same evolutionary forces that shape all DNA replicating within a cell, meaning that they will be subject to selection and may accrue mutations that affect their function, especially if doing so improves the fitness of the host cell. In Chapter 1 I reflect on the history of engineered plasmids and the inherent conflicts between engineered plasmids and their host bacteria. In Chapter 2 I describe pLannotate, a webserver and command line tool that is designed specifically for annotating engineered DNA and plasmids. I use this tool to demonstrate that most engineered plasmids contain fragments of functional genetic elements that are hidden relics from their construction histories. In Chapter 3 I develop a method for identifying variants of common functional elements used in engineered plasmids that likely arose multiple times independently by convergent engineering or evolution. I compile a list of hundreds of these undocumented variants and others that are found in plasmids from many labs. In Chapter 4 I analyze the ColE1 origin of replication, which is the most common functional element in engineered plasmids for evidence of unintended evolution. I show that a majority of convergent ColE1 variants have significantly altered copy numbers, which could be due to selection to reduce burden in some cases. In Chapter 5 I summarize lessons from each of the previous chapters, discuss caveats, and offer suggestions for future research in these areas.