Assembly of Highly Standardized Gene Fragments for High-Level Production of Porphyrins in E. coli

Morten Thrane Nielsen, Karina Marie Madsen, Susanna Seppala, Ulla Christensen, Lone Riisberg, Scott James Harrison, Birger Lindberg Møller, Morten Nørholm

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Standardization of molecular cloning greatly facilitates advanced DNA engineering, parts sharing, and collaborative efforts such as the iGEM competition. All of these attributes facilitate exploitation of the wealth of genetic information made available by genome and RNA sequencing. Standardization also comes at the cost of reduced flexibility. We addressed this paradox by formulating a set of design principles aimed at maximizing standardization while maintaining high flexibility in choice of cloning technique and minimizing the impact of standard sequences. The design principles were applied to formulate a molecular cloning pipeline and iteratively assemble and optimize a six-gene pathway for protoporphyrin IX synthesis in Escherichia coli. State of the art production levels were achieved through two simple cycles of engineering and screening. The principles defined here are generally applicable and simplifies the experimental design of projects aimed at biosynthetic pathway construction or engineering.
Original languageEnglish
JournalA C S Synthetic Biology
Volume4
Issue number3
Pages (from-to)274-282
Number of pages9
ISSN2161-5063
DOIs
Publication statusPublished - 2015

Keywords

  • Molecular cloning
  • Biobricks
  • Synthetic biology
  • Uracil-excision
  • Cloning
  • Porphyrin bioengineering

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