CRISPR/Cas9-based engineering of Escherichia coli for biobrick-based minicircles
• Gabriel A. Monteiro, Professor @ IST/ULisboa, PT
• Pedro M. Paulo, Research Scientist @ IST/ULisboa, PT
Gene therapy and DNA vaccination are fields of study in which researchers have a high interest, being registered an ongoing increase in reported clinical trials. Plasmid DNA as a non-viral delivery vector has been widely applied, despite its low transfection efficiency and transgene expression. The pursue of safer and more efficient delivery vectors resulted in the development of an in vivo recombination process in Escherichia coli in which a vector comprising only an eukaryotic sequence is obtained by excision from a replicative and prokaryotic sequence containing plasmid – the minicircle DNA production system. This results in the formation, during bacterial cellular growth, of a therapeutically valuable DNA molecule – the minicircle – and of a miniplasmid comprised of the prokaryotic sequence. However, the low pDNA specific yield of existing minicircle-producing E. coli strains calls for the development of a strain improved for minicircle production. The preliminary proposed research program will rely on a synthetic biology approach and can be divided into three tasks: (i) optimization of parA resolvase expression, (ii) construction of an Escherichia coli strain improved for minicircle production and process scale-up and (iii) development of a standard parental plasmid for on-demand minicircle construction and evaluation of eukaryotic promoters and polyadenylation signals on gene expression.
Silva-Santos AR, Alves CPA, Monteiro GA, Azevedo AM, Prazeres DMF, “Multimodal chromatography of supercoiled minicircles: a closer look into DNA-ligand interactions”, Separation and Purification Technology, 212, 161-170, 2019
Serra J, Alves CPA, Brito L, Monteiro GA, Cabral JMS, Prazeres DMF, da Silva CL, “Engineering of human mesenchymal stem/stromal cells (MSC) with VEGF-encoding minicircles for angiogenic ex vivo gene therapy”, Human Gene Therapy 30(3):316-329, 2018
Alves CPA, Simčíková M, Brito L, Monteiro GA, Prazeres DMF, “Production and Purification of Supercoiled Minicircles by a Combination of In Vitro Endonuclease Nicking and Hydrophobic Interaction Chromatography”, Human Gene Therapy Methods 29(4):157-168, 2018.
Alves C, Prazeres DM, Reisch C, Monteiro G, “Improving minicircle DNA production in E. coli – in vivo analysis of recombination efficiency after re-design of ParA resolvase 5’-UTR”, MicroBiotec2019 December 5-7, 2019, Coimbra, Portugal
Alves CPA, Šimčíková M, Prazeres DMF, Monteiro GA, ”Optimization of ParA resolvase untranslated region – a strategy to boost minicircle DNA production”, MicroBiotec2017 December 7-8, 2017, Porto, Portugal
Alves CPA, Prazeres DMF, Monteiro GA, ”Escherichia coli Genome Engineering for Improved Minicircle Production”, PhD open days 2018Instituto Superior Técnico, Lisboa, Portugal
Cláudia P.A. Alves, Duarte Miguel F. Prazeres, Gabriel Monteiro, ”Improving minicircle DNA production by in silico re-design of the ParA resolvase untranslated region », 12th ESBES – European Symposium on Biochemical Engineering Sciences, September 2018, Lisboa, Portugal
DOCTORAL PROGRAM (36 ECTS)
• General Doctoral Training (6 ECTS)
• Advanced Experimental Techniques and methodologies (6 ECTS)
• Bioentrepreneurship (6 ECTS)
• Outreach and Teaching Skills (6 ECTS)
• Gene Therapy (6 ECTS)
• Steam cell engineering(6 ECTS)
• Animal Cell Technology course, organized by the European Society of Animal Cell Technology in Llafranc, Girona, Spain, from 24th to 28th September 2017 (30 hours)
• Cell Culture-based Viral Vaccines course – 3rd edition, organized by the European Society for Animal Cell Technology in Llafranc, Girona, Spain from 18-22 September 2018.