TARSyn: Tunable Antibiotic Resistance Devices Enabling Bacterial Synthetic Evolution and Protein Production

Maja Rennig, Virginia Martinez, Kiavash Mirzadeh, Finn Dunas, Belinda Röjsäter, Daniel O Daley, Morten Nørholm*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

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.
Original languageEnglish
JournalA C S Synthetic Biology
Volume7
Issue number2
Pages (from-to)432-442
Number of pages11
ISSN2161-5063
DOIs
Publication statusPublished - 2018

Keywords

  • Antibiotic resistance
  • Protein production
  • Selection system
  • Synthetic evolution
  • Translation initiation region
  • Translational coupling

Cite this

Rennig, Maja ; Martinez, Virginia ; Mirzadeh, Kiavash ; Dunas, Finn ; Röjsäter, Belinda ; Daley, Daniel O ; Nørholm, Morten. / TARSyn: Tunable Antibiotic Resistance Devices Enabling Bacterial Synthetic Evolution and Protein Production. In: A C S Synthetic Biology. 2018 ; Vol. 7, No. 2. pp. 432-442.
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abstract = "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.",
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TARSyn: Tunable Antibiotic Resistance Devices Enabling Bacterial Synthetic Evolution and Protein Production. / Rennig, Maja; Martinez, Virginia; Mirzadeh, Kiavash; Dunas, Finn; Röjsäter, Belinda; Daley, Daniel O; Nørholm, Morten.

In: A C S Synthetic Biology, Vol. 7, No. 2, 2018, p. 432-442.

Research output: Contribution to journalJournal articleResearchpeer-review

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AU - Rennig, Maja

AU - Martinez, Virginia

AU - Mirzadeh, Kiavash

AU - Dunas, Finn

AU - Röjsäter, Belinda

AU - Daley, Daniel O

AU - Nørholm, Morten

PY - 2018

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