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TARSyn: Tunable Antibiotic Resistance Devices Enabling Bacterial Synthetic Evolution and Protein Production
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. CloneOpt AB, Sweden.
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Number of Authors: 7
2018 (English)In: ACS Photonics, ISSN 2186-2311, E-ISSN 2161-5063, Vol. 7, no 2, p. 432-442Article in journal (Refereed) Published
Abstract [en]

Evolution can be harnessed to optimize synthetic biology designs. A prominent example is recombinant protein production-a 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 expression-an 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.

Place, publisher, year, edition, pages
2018. Vol. 7, no 2, p. 432-442
Keyword [en]
protein production, antibiotic resistance, translational coupling, selection system, synthetic evolution, translation initiation region
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-154620DOI: 10.1021/acssynbio.7b00200ISI: 000426012600016PubMedID: 29257878OAI: oai:DiVA.org:su-154620DiVA, id: diva2:1194621
Available from: 2018-04-03 Created: 2018-04-03 Last updated: 2018-04-03Bibliographically approved

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Mirzadeh, KiavashDaley, Daniel O.
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