Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase
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 pa...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Springer Berlin Heidelberg
2016
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| Online Access: | http://hdl.handle.net/1721.1/104377 https://orcid.org/0000-0003-0437-3157 |
| _version_ | 1826188737490452480 |
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| author | Brito, Liliana Alves, Cláudia P. A. Monteiro, Gabriel A. Simcikova, Michaela Jones, Kristala L Prazeres, Duarte Miguel Jones, Kristala L. |
| author2 | MIT-Portugal Program |
| author_facet | MIT-Portugal Program Brito, Liliana Alves, Cláudia P. A. Monteiro, Gabriel A. Simcikova, Michaela Jones, Kristala L Prazeres, Duarte Miguel Jones, Kristala L. |
| author_sort | Brito, Liliana |
| collection | MIT |
| description | 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 P[subscript BAD]/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. |
| first_indexed | 2024-09-23T08:04:16Z |
| format | Article |
| id | mit-1721.1/104377 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T08:04:16Z |
| publishDate | 2016 |
| publisher | Springer Berlin Heidelberg |
| record_format | dspace |
| spelling | mit-1721.1/1043772022-09-30T07:16:04Z Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase Brito, Liliana Alves, Cláudia P. A. Monteiro, Gabriel A. Simcikova, Michaela Jones, Kristala L Prazeres, Duarte Miguel Jones, Kristala L. MIT-Portugal Program Massachusetts Institute of Technology. Department of Chemical Engineering Monteiro, Gabriel A. Simcikova, Michaela Jones, Kristala L. Prazeres, Duarte Miguel 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 P[subscript BAD]/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. MIT-Portugal Program Fundação para a Ciência e a Tecnologia (PhD grant SFRH/BD/33786/2009) 2016-09-22T22:14:03Z 2017-03-01T16:14:47Z 2016-05 2016-04 2016-08-18T15:24:36Z Article http://purl.org/eprint/type/JournalArticle 0175-7598 1432-0614 http://hdl.handle.net/1721.1/104377 Šimčíková, Michaela et al. “Improvement of DNA Minicircle Production by Optimization of the Secondary Structure of the 5′-UTR of ParA Resolvase.” Applied Microbiology and Biotechnology 100.15 (2016): 6725–6737. https://orcid.org/0000-0003-0437-3157 en http://dx.doi.org/10.1007/s00253-016-7565-x Applied Microbiology and Biotechnology Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ Springer-Verlag Berlin Heidelberg application/pdf Springer Berlin Heidelberg Springer Berlin Heidelberg |
| spellingShingle | Brito, Liliana Alves, Cláudia P. A. Monteiro, Gabriel A. Simcikova, Michaela Jones, Kristala L Prazeres, Duarte Miguel Jones, Kristala L. Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase |
| title | Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase |
| title_full | Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase |
| title_fullStr | Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase |
| title_full_unstemmed | Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase |
| title_short | Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase |
| title_sort | improvement of dna minicircle production by optimization of the secondary structure of the 5 utr of para resolvase |
| url | http://hdl.handle.net/1721.1/104377 https://orcid.org/0000-0003-0437-3157 |
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