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Registry in a tube: multiplexed pools of retrievable parts for genetic design space exploration.

Nucleic acids research (2016-12-23)
Lauren B A Woodruff, Thomas E Gorochowski, Nicholas Roehner, Tarjei S Mikkelsen, Douglas Densmore, D Benjamin Gordon, Robert Nicol, Christopher A Voigt
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
M9, Minimal Salts, 5X, powder, minimal microbial growth medium
Supelco
LB broth, Miller, Microbiologically tested

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