Publicação
The role of the restriction-modification system of Clostridium pasteurianum on its electro-transformation
| Resumo: | Clostridium pasteurianum is a Gram-positive and anaerobic bacterium with a great biotechnological potential. It is one of the few microorganisms capable of hydrolyzing glycerol to produce solvents as ethanol and butanol, which have a wide applicability in the market as biofuels. The development of a genetic system for this microorganism would increase its application opportunities since gene overexpression or inactivation could improve their solventogenic characteristics. Its genetic information is already known but this organism has a particular resistance to transformation. This resistance can be explained by a very efficient restriction system that does not allow the entrance of non-methylated DNA or DNA with a methylation pattern different from it. Therefore, foreign DNA must be correctly methylated prior to transformation. For this purpose, a specific methyltransferase is needed to transfer methyl groups to a certain nucleotide of a specific sequence. The goal of this thesis was to create a genetic system in C. pasteurianum that allows genome modification and foreign protein expression, ultimately improving C. pasteurianum DSM 525 transformation. Preliminary simple electro-transformations in which the parameters to make competent cells and the electroporation conditions were altered, did not result in positive results. Being aware of the possibility of a restriction system presence in this organism, experiments with M.MspI methylated DNA were performed, however they demonstrated the inability of this methyltransferase to improve the microorganism transformation. The presence of restriction enzymes was confirmed when a characterization of the restriction system of C. pasteurianum was performed using MspI methylated and non-methylated DNA. The presence of a discrete digestion pattern was detected, and M.MspI methylation could not protect the foreign DNA from C. pasteurianum restriction action. The polyamine spermidine, with known affinity for negatively charged DNA, showed to be efficient against C. pasteurianum crude extract digestion action, however not sufficiently to facilitate this microorganism electrotransformation. By accessing the genome information, the Restriction/Modification (R/M) systems of this microorganism were analyzed. The GATC type IIP R/M system was chosen in order to verify the restriction and methylation enzymes activity with the same target sequence. Three genes, one REase (DpnII) and two MTases (Dam and MdpnII) were cloned in pETduet-1, followed by overproduction in BL21 (DE3). The codon usage of the host and original organism were not compatible, and the protein production in tRNAs provider strains was tested. Protein production was detected, however was not possible to re-confirm their presence. The common protein folding problems were analyzed using a disulfide bond enhancer strain. Nevertheless, the production problem may not be related to this, since no different protein over-production was detected. Restriction reactions with the REase BstUI and C. pasteurianum crude extract, using DNA methylated by M.SssI (m5CG), were developed and showed that the REase responsible for hindering foreign DNA entering C. pasteurianum recognizes the sequence 5'-CGCG- 3'. In a second analysis of the C. pasteurianum genome a methyltransferase-encoding gene was identified that may be involved in methylating the sequence 5'-CGCG- 3'. The in silico analysis was performed and its codon usage was also improved to be compatible with E. coli. In this work, the reasons for C. pasteurianum’s recalcitrance to transformation were identified, the knowledge about its R/M systems was extended, and a proposal to efficiently transform this bacterium was provided. |
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| Autores principais: | Magalhães, Carla Isabel Pereira |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Clostridium pasteurianum is a Gram-positive and anaerobic bacterium with a great biotechnological potential. It is one of the few microorganisms capable of hydrolyzing glycerol to produce solvents as ethanol and butanol, which have a wide applicability in the market as biofuels. The development of a genetic system for this microorganism would increase its application opportunities since gene overexpression or inactivation could improve their solventogenic characteristics. Its genetic information is already known but this organism has a particular resistance to transformation. This resistance can be explained by a very efficient restriction system that does not allow the entrance of non-methylated DNA or DNA with a methylation pattern different from it. Therefore, foreign DNA must be correctly methylated prior to transformation. For this purpose, a specific methyltransferase is needed to transfer methyl groups to a certain nucleotide of a specific sequence. The goal of this thesis was to create a genetic system in C. pasteurianum that allows genome modification and foreign protein expression, ultimately improving C. pasteurianum DSM 525 transformation. Preliminary simple electro-transformations in which the parameters to make competent cells and the electroporation conditions were altered, did not result in positive results. Being aware of the possibility of a restriction system presence in this organism, experiments with M.MspI methylated DNA were performed, however they demonstrated the inability of this methyltransferase to improve the microorganism transformation. The presence of restriction enzymes was confirmed when a characterization of the restriction system of C. pasteurianum was performed using MspI methylated and non-methylated DNA. The presence of a discrete digestion pattern was detected, and M.MspI methylation could not protect the foreign DNA from C. pasteurianum restriction action. The polyamine spermidine, with known affinity for negatively charged DNA, showed to be efficient against C. pasteurianum crude extract digestion action, however not sufficiently to facilitate this microorganism electrotransformation. By accessing the genome information, the Restriction/Modification (R/M) systems of this microorganism were analyzed. The GATC type IIP R/M system was chosen in order to verify the restriction and methylation enzymes activity with the same target sequence. Three genes, one REase (DpnII) and two MTases (Dam and MdpnII) were cloned in pETduet-1, followed by overproduction in BL21 (DE3). The codon usage of the host and original organism were not compatible, and the protein production in tRNAs provider strains was tested. Protein production was detected, however was not possible to re-confirm their presence. The common protein folding problems were analyzed using a disulfide bond enhancer strain. Nevertheless, the production problem may not be related to this, since no different protein over-production was detected. Restriction reactions with the REase BstUI and C. pasteurianum crude extract, using DNA methylated by M.SssI (m5CG), were developed and showed that the REase responsible for hindering foreign DNA entering C. pasteurianum recognizes the sequence 5'-CGCG- 3'. In a second analysis of the C. pasteurianum genome a methyltransferase-encoding gene was identified that may be involved in methylating the sequence 5'-CGCG- 3'. The in silico analysis was performed and its codon usage was also improved to be compatible with E. coli. In this work, the reasons for C. pasteurianum’s recalcitrance to transformation were identified, the knowledge about its R/M systems was extended, and a proposal to efficiently transform this bacterium was provided. |
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