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. Each pool is built via multiplexed assembly and sequenced using a customized version of targeted next-generation sequencing. Each pool consists of ~20 Mb of up to 5,000 unique and sequence-perfect 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 constructed. 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 that can then be efficiently re-accessed for guided combinatorial search.
- Bristol BioDesign Institute
- polymerase chain reaction genes libraries nitrogen fixation oncogenes genetics transcriptional repression massively-parallel genome sequencing verification
- synthetic biology