title: Registry in a tube: multiplexed pools of retrievable parts for genetic design space exploration.
abstract: Genetic designs can consist of dozens of genes and hundreds of genetic parts. After evaluating a design, it is desirable to implement changes without the cost and burden of starting the construction process from scratch. Here, we report a two-step process where a large design space is divided into deep pools of composite parts, from which individuals are retrieved and assembled to build a final construct. The pools are built via multiplexed assembly and sequenced using next-generation sequencing. Each pool consists of ∼20 Mb of up to 5000 unique and sequence-verified composite parts that are barcoded for retrieval by PCR. This approach is applied to a 16-gene nitrogen fixation pathway, which is broken into pools containing a total of 55 848 composite parts (71.0 Mb). The pools encompass an enormous design space (1043 possible 23 kb constructs), from which an algorithm-guided 192-member 4.5 Mb library is built. Next, all 1030 possible genetic circuits based on 10 repressors (NOR/NOT gates) are encoded in pools where each repressor is fused to all permutations of input promoters. These demonstrate that multiplexing can be applied to encompass entire design spaces from which individuals can be accessed and evaluated.
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.|Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
authors.forename: Lauren B A
authors.initials: LBA
authors.lastname: Woodruff
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.|Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Gorochowski
authors.affiliation: Biological Design Center, Department of Electrical and Computer Engineering, Boston University, Boston, MA, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Roehner
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.
authors.forename: Tarjei S
authors.initials: TS
authors.lastname: Mikkelsen
authors.affiliation: Biological Design Center, Department of Electrical and Computer Engineering, Boston University, Boston, MA, USA.
authors.forename: Douglas
authors.initials: D
authors.lastname: Densmore
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.|Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
authors.forename: D Benjamin
authors.initials: DB
authors.lastname: Gordon
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Nicol
authors.affiliation: Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA.|Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Voigt
mesh_terms: D000465:Algorithms; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D053263:Gene Regulatory Networks; D005818:Genetic Engineering / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D007709:Klebsiella / Q000235:genetics / Q000378:metabolism; D009586:Nitrogen Fixation / Q000235:genetics; D009591:Nitrogenase / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic
chemical_list: D009591:Nitrogenase