on 06-13-201408:10 AM - edited on 10-15-202111:22 AM by AnalyteGuru
Nielsen MT, Madsen KM, Seppälä S, Christensen U, Riisberg L, Harrison SJ, Møller BL, Nørholm MH. ACS Synth Biol. 2014 Jun 6. Standardization of molecular cloning greatly facilitates advanced DNA engineering, parts sharing and collaborative efforts like 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 maximal standardization without compromising flexibility in choice of cloning technique. 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.