A Cell-Free Transcription-Translation Kit (MYtxtl®) for Rapid Prototyping of Transcription Units Assembled from Error-Free Modular Parts

Rapid prototyping of genetic circuitry requires the ability to assemble and test different combinations of modular, error-free DNA parts in a high throughput manner. We describe here a modular parts system for rapidly assembling and testing linear DNA transcription units in an in vitro cell-free transcription-translation system (MYtxtl®) derived from E. coli. This modular parts system consists of a collection of separate promoters, open reading frames and terminators that can be combined on demand into transcription units that can be assembled and tested in the same day.

We synthesized and sequence-verified several plasmids that each contains one modular part. As a demonstration of rapid prototyping, we assembled multiple transcription units in parallel on a microplate using one plasmid each of a promoter, open reading frame, and terminator. These plasmids were digested and assembled in a directional Golden Gate cloning reaction into linear transcription units. This linear template was then PCR amplified, purified and added to the MYtxtl^® reaction. Linear templates were incubated for as short as 4 hours followed by measurement of mRNA synthesis and/or protein production. This allowed us to compare the expression of a particular protein under the control of promoters of different strengths in one work day.

One limitation of using linear DNA molecules as templates in MYtxtl^® reactions is their susceptibility to rapid nuclease degradation by the RecBCD enzyme and other nucleases present in the MYtxtl^® extract. We therefore used direct modifications of the linear DNA templates and nuclease inhibitors added to the reaction to increase protection of templates from degradation. These highly efficient and cost-effective methods increased protection of linear DNA templates from degradation by several fold relative to reactions using templates without modifications or nuclease inhibitors. This allowed for protein production from linear templates that approached that of circular plasmids, eliminating the need to spend time cloning genetic circuits.

Starting from a plasmid library of modular parts, we successfully assembled and tested multiple transcription units within an 8 to 24 hour period. These reactions can be used to analyze transcription, translation, gene circuitry and protein function (with the additional step of protein purification). This rapid assembly and testing of modular parts in the MYtxtl^® system allows for high-throughput prototyping of a diverse number of transcription units and proteins, making the MYtxtl^® system a valuable tool to synthetic biologists.