Journal article
TARSyn: Tunable Antibiotic Resistance Devices Enabling Bacterial Synthetic Evolution and Protein Production
Novo Nordisk Foundation Center for Biosustainability1
Center for Biomembrane Research, Department of Biochemistry and Biophysics2
19468 Upplands Väsby3
17169 Solna4
Evolution can be harnessed to optimize synthetic biology designs. A prominent example is recombinant protein productiona dominating theme in biotechnology for more than three decades. Typically, a protein coding sequence (cds) is recombined with genetic elements, such as promoters, ribosome binding sites and terminators, which control expression in a cell factory.
A major bottleneck during production is translational initiation. Previously we identified more effective translation initiation regions (TIRs) by creating sequence libraries and then selecting for a TIR that drives high-level expressionan example of synthetic evolution. However, manual screening limits the ability to assay expression levels of all putative sequences in the libraries.
Here we have solved this bottleneck by designing a collection of translational coupling devices based on a RNA secondary structure. Exchange of different sequence elements in this device allows for different coupling efficiencies, therefore giving the devices a tunable nature. Sandwiching these devices between the cds and an antibiotic selection marker that functions over a broad dynamic range of antibiotic concentrations adds to the tunability and allows expression levels in large clone libraries to be probed using a simple cell survival assay on the respective antibiotic. The power of the approach is demonstrated by substantially increasing production of two commercially interesting proteins, a Nanobody and an Affibody.
The method is a simple and inexpensive alternative to advanced screening techniques that can be carried out in any laboratory.
Language: | English |
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Publisher: | American Chemical Society |
Year: | 2018 |
Pages: | 432-442 |
ISSN: | 21615063 |
Types: | Journal article |
DOI: | 10.1021/acssynbio.7b00200 |