An inability to reliably predict quantitative behaviors for novel combinations of genetic elements limits the rational engineering of biological systems. We developed an expression cassette architecture for genetic elements controlling transcription and translation initiation in Escherichia coli: transcription elements encode a common mRNA start, and translation elements use an overlapping genetic motif found in many natural systems. We engineered libraries of constitutive and repressor-regulated promoters along with translation initiation elements following these definitions. We measured activity distributions for each library and selected elements that collectively resulted in expression across a 1,000-fold observed dynamic range. We studied all combinations of curated elements, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with ∼93% reliability. We expect the genetic element definitions validated here can be collectively expanded to create collections of public-domain standard biological parts that support reliable forward engineering of gene expression at genome scales.
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We thank C. Smolke for discussions. We acknowledge support from a US National Science Foundation grant to the BIOFAB (EEC 0946510) and unrestricted gifts from Genencor, Agilent and DSM. J.C.G. acknowledges financial support from the Portuguese Fundação para a Ciência e a Tecnologia (FCT) (SFRH/BD/47819/2008); G.C. acknowledges the Human Frontier Science Program (LT000873/2011-l) and Bettencourt Schueller Foundation; A.P.A. and D.E. acknowledge the Synthetic Biology Engineering Research Center under National Science Foundation grant 04-570/0540879. This work was conducted at the Joint BioEnergy Institute supported by the Office of Science, Office of Biological and Environmental Research, US Department of Energy, contract DE-AC02-05CH11231.
The authors declare no competing financial interests.
Supplementary Figures 1–32, Supplementary Table 1 and Supplementary Note (PDF 7374 kb)
List of parts, plasmids and strains used in the present work. Columns as follows: A, number; B, vector backbone; C, abstract part number for promoter element, indicated as “apFAB#”; D, promoter name; E, abstract part number for 5' UTR element, indicated as “apFAB#”; F, 5' UTR name used in the main text; G, abstract part number for GOI element, indicated as “apFAB#”; H, GOI name; I, plasmid number “pFAB#”; J, antibiotics; K, replication origin; L, strain; M, strain number “sFAB#”; N, project name. (XLS 462 kb)
List of primers used in the present work. Columns as follows: A, number; B, oligonucleotide number (“oFAB#”; primers used for sequencing are denoted as “soFAB#”); C, forward and reverse primers are indicated as FW and RV; D, information notes for the primer; E, primer sequence (5' to 3'); F, project name. (XLS 129 kb)
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Mutalik, V., Guimaraes, J., Cambray, G. et al. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat Methods 10, 354–360 (2013). https://doi.org/10.1038/nmeth.2404
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