Skip to main content

Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase

Abstract

The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, we focus on the 5′ untranslated region (5′-UTR) of parA messenger RNA (mRNA). The arabinose-inducible PBAD/araC promoter controls ParA expression and strains with improved arabinose uptake are used. The 27-nucleotide-long 5′-UTR of parA mRNA was optimized using a predictive thermodynamic model. An analysis of original and optimized mRNA subsequences predicted a decrease of 8.6–14.9 kcal/mol in the change in Gibbs free energy upon assembly of the 30S ribosome complex with the mRNA subsequences, indicating a more stable mRNA-rRNA complex and enabling a higher (48–817-fold) translation initiation rate. No effect of the 5′-UTR was detected when ParA was expressed from a low-copy number plasmid (∼14 copies/cell), with full recombination obtained within 2 h. However, when the parA gene was inserted in the bacterial chromosome, a faster and more effective recombination was obtained with the optimized 5′-UTR. Interestingly, the amount of this transcript was 2.6–3-fold higher when compared with the transcript generated from the original sequence, highlighting that 5′-UTR affects the level of the transcript. A Western blot analysis confirmed that E. coli synthesized higher amounts of ParA with the new 5′-UTR (∼1.8 ± 0.7-fold). Overall, these results show that the improvements made in the 5′-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

Download references

Acknowledgments

This work was supported by the MIT-Portugal Program, Fundação para a Ciência e a Tecnologia (grant UID/BIO/04565/2013 awarded to iBB—Institute for Bioengineering and Biosciences and PhD grant SFRH/BD/33786/2009 to Michaela Šimčíková).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Gabriel A. Monteiro.

Ethics declarations

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

ESM 1

(PDF 616 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Šimčíková, M., Alves, C.P.A., Brito, L. et al. Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase. Appl Microbiol Biotechnol 100, 6725–6737 (2016). https://doi.org/10.1007/s00253-016-7565-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-016-7565-x

Keywords

  • Minicircle
  • parA resolvase
  • 5′-UTR
  • Ribosome binding site
  • mRNA secondary structure
  • Translational initiation rate