Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
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.
Show others and affiliations
Number of Authors: 72018 (English)In: ACS Photonics, E-ISSN 2330-4022, 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
Keywords [en]
protein production, antibiotic resistance, translational coupling, selection system, synthetic evolution, translation initiation region
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
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: 2018-08-06Bibliographically approved
In thesis
1. Engineering microbial cell factories for protein production
Open this publication in new window or tab >>Engineering microbial cell factories for protein production
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are often produced using microbial cell factories for academic or industrial purposes. Protein production is however not an open-and-shut procedure. Production yields often vary in an unpredictable and context dependent manner, limiting the rational design of a straightforward production experiment.

This thesis gives an overview of how proteins are biosynthesised in bacterial cells and how this knowledge is used to produce proteins recombinantly in a host organism such as Escherichia coli. In the present investigation, we reason that unpredictable and poor protein production yields could result from incompatibility between the vector derived 5’ UTR and the 5’ end of the cloned CDS which leads to an unevolved translation initiation region (TIR). Data presented in this thesis show that an unevolved TIR could work more efficiently and yield more produced protein if subjected to synthetic evolution. Clones with an engineered synthetically evolved TIR showed enhanced protein production in both small- and large-scale production setups. This engineering method could lower production expenses, which in turn would result in increased functional determination of proteins and expanded availability of protein-based medicine to people globally. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. p. 60
Keywords
Protein production, expression vector, recombinant DNA, Translation initiation region, Escherichia coli, mRNA secondary structure, Synthetic evolution
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-158482 (URN)978-91-7797-366-9 (ISBN)978-91-7797-367-6 (ISBN)
Public defence
2018-09-21, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2018-08-29 Created: 2018-08-06 Last updated: 2018-08-29Bibliographically approved

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Mirzadeh, KiavashDaley, Daniel O.
By organisation
Department of Biochemistry and Biophysics
In the same journal
ACS Photonics
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 14 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf