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

## TL;DR

Researchers developed a low-cost method to synthesize genes from DNA building blocks, creating a glowing reporter for studying plant gene activity.

## Contribution

A new pipeline called iggypop enables rapid, low-cost synthesis of large DNA constructs, including a single-transcript bioluminescent reporter for plant research.

## Key findings

- The iggypop pipeline allows efficient synthesis of hundreds of genes from oligonucleotide pools.
- STARBURST reporters, combining five bioluminescence genes in a single transcript, produce strong luminescence in plants.
- GC content significantly impacts gene expression in plant synthetic biology.

## Abstract

Large-scale DNA synthesis from oligonucleotide pools is valuable for synthetic biology as it enables low-cost gene synthesis. Here, we developed a simple computational–experimental pipeline, iggypop, that enables rapid gene synthesis and exploration of design space, a critical need in plant synthetic biology. We used it to build a series of single-transcript autobioluminescent reporters (STARBURSTs) that link the five enzymes of a fungal bioluminescence pathway in a single transcript using LP4/2A linkers. The low cost and ease of iggypop allowed us to rapidly test several genetic designs to yield a bright reporter for facile measurements of plant gene expression and illuminated an unappreciated role of GC content in plant synthetic gene design. Thus, iggypop accelerates the development of new genetic parts.

Methods for fast and inexpensive gene synthesis from oligonucleotide pools enable rapid iteration of genetic designs. Here, we describe iggypop (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 iggypop 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 Nicotiana benthamiana assays. Based on this observation, we synthesized six GC-boosted STARBURSTs, which produced robust luminescence in both transient assays and transgenic Arabidopsis plants. Thus, iggypop enabled the rapid synthesis of multiple genetic designs to deliver a bright single transcript autobioluminescent reporter. Iggypop should enable the facile synthesis and optimization of new genetic parts and complex polycistronic pathways.