Synthesis of large single-transcript pathways from oligonucleotide pools: Design of STARBURST, an autobioluminescent reporter.

Abstract

Methods for fast and inexpensive gene synthesis from oligonucleotide pools enable rapid iteration of genetic designs. Here, we describe <i>iggypop</i> (indexed Golden Gate gene assembly from PCR-amplified oligonucleotide pools), a simple computational-experimental pipeline that allows for low-cost design and synthesis of hundreds of genes from oligonucleotide pools using Golden Gate assembly methods. We used <i>iggypop</i> to synthesize a series of single-transcript autonomously bioluminescent reporters (STARBURSTs) that link the five genes of a fungal bioluminescence pathway via ribosomal skipping LP4/2A sequences into a 9.5 kb transcript that function in planta. We also synthesized RUBY reporters (a reporter gene system producing red betalain pigment) recoded to match dicot codon usage, as RUBY was codon optimized for rice codon usage and has a high GC content. Surprisingly, the recoded RUBYs substantially reduced betalain production in transient <i><i>Nicotiana</i> benthamiana</i> assays. Based on this observation, we synthesized six GC-boosted STARBURSTs, which produced robust luminescence in both transient assays and transgenic <i>Arabidopsis</i> plants. Thus, <i>iggypop</i> enabled the rapid synthesis of multiple genetic designs to deliver a bright single transcript autobioluminescent reporter. <i>Iggypop</i> should enable the facile synthesis and optimization of new genetic parts and complex polycistronic pathways.