Abstract
Directed laboratory evolution is a common technique to obtain an evolved bacteria strain with a desired phenotype. This technique is especially useful as a supplement to rational engineering for complex phenotypes such as increased biocatalyst tolerance to toxic compounds. However, reverse engineering efforts are required in order to identify the mutations that occurred, including single nucleotide polymorphisms (SNPs), insertions/deletions (indels), duplications, and rearrangements. In this protocol, we describe the steps to (1) obtain and sequence the genomic DNA, (2) process and analyze the genomic DNA sequence data, and (3) verify the mutations by Sanger resequencing.
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Acknowledgement
We would like to thank Michael Baker at the DNA facility of the Iowa State University Office of Biotechnology for his input on next-generation sequencing using the Illumina platform. We also thank Emily Rickenbach, an undergraduate student who helped automate the method for picking primers for verifying mutations. Funding was provided for this work by the NSF Engineering Research Center for Biorenewable Chemicals (CBiRC), NSF award number EEC-0813570 and NSF Energy for Sustainability award number CBET-1133319.
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Royce, L., Boggess, E., Jin, T., Dickerson, J., Jarboe, L. (2013). Identification of Mutations in Evolved Bacterial Genomes. In: Alper, H. (eds) Systems Metabolic Engineering. Methods in Molecular Biology, vol 985. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-299-5_13
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DOI: https://doi.org/10.1007/978-1-62703-299-5_13
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