Cloning of affecting pyruvate decarboxylase gene in the production bioethanol of agricultural waste in the E.coli bacteria
Introduction: Ethanol made by a biomass is one of the useful strategies in terms of economic and environmental and as a clean and safe energy to replace fossil fuels considered and examined. Materials and methods: In this study, key enzyme in the production of ethanol (Pyruvate decarboxylase) from...
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University of Isfahan
2016-09-01
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| Series: | Biological Journal of Microorganism |
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| Online Access: | http://uijs.ui.ac.ir/bjm/browse.php?a_code=A-10-545-1&slc_lang=en&sid=1 |
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| author | Masome Zeinali Bahman Hosseini Soodabeh Jahanbakhsh Mahmod Rezazadeh bari Meisam Tabatabaee |
| author_facet | Masome Zeinali Bahman Hosseini Soodabeh Jahanbakhsh Mahmod Rezazadeh bari Meisam Tabatabaee |
| author_sort | Masome Zeinali |
| collection | DOAJ |
| description | Introduction: Ethanol made by a biomass is one of the useful strategies in terms of economic and environmental and as a clean and safe energy to replace fossil fuels considered and examined.
Materials and methods: In this study, key enzyme in the production of ethanol (Pyruvate decarboxylase) from Zymomonas mobilis bacteria was isolated and cloned at E. coli bacteria by freeze and thaw method. For gene cloning, we used specific primers of pdc and PCR reaction and then pdc gene isolated and pET 28a plasmid double digested with (Sal I and Xho I) enzymes. Digestion Products were ligated by T4 DNA ligase in 16 °C for 16 hours.
Results: Results of bacteria culture showed that a few colonies containing pET 28a plasmid could grow. Result of colony pcr of pdc gene with specific primers revealed 1700 bp bands in 1% agarose gel electrophoresis. The results of PCR with T7 promotor forward primer and pdc revers primer have proved the accurate direction of integration of pdc gene into plasmid and revealed 1885 bp band. Double digestion of recombinant plasmid with SalI and XhoI enzymes revealed same bands. Finally, RT showed the expected band of 1700 bp that implies the desired gene expression in the samples.
Discussion and conclusion: Due to the increased production of ethanol via pyruvate decarboxylase gene cloning in expression plasmids with a strong promoter upstream of the cloning site can conclude that, pyruvate decarboxylase cloning as a key gene would be useful and according to beneficial properties of E. coli bacteria, transfering the gene to bacteria appears to be reasonable. |
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| format | Article |
| id | doaj.art-428da65ef01c4ca0acce8aa7047273e8 |
| institution | Directory Open Access Journal |
| issn | 2322-5173 2322-5181 |
| language | English |
| last_indexed | 2024-12-20T04:04:34Z |
| publishDate | 2016-09-01 |
| publisher | University of Isfahan |
| record_format | Article |
| series | Biological Journal of Microorganism |
| spelling | doaj.art-428da65ef01c4ca0acce8aa7047273e82022-12-21T19:54:04ZengUniversity of IsfahanBiological Journal of Microorganism2322-51732322-51812016-09-015181128Cloning of affecting pyruvate decarboxylase gene in the production bioethanol of agricultural waste in the E.coli bacteriaMasome Zeinali0Bahman Hosseini1Soodabeh Jahanbakhsh2Mahmod Rezazadeh bari3Meisam Tabatabaee4 M.Sc. of Agricultural biotechnology, University of Mohaghegh Ardabili, Ardabil, Iran Associate Professor of Plant Biotechnology of Agriculture College and Institute of Biotechnology of Urmia University, Iran Associate Professor of Plant Breeding, University of Mohaghegh Ardabili, Ardabil, Iran Associate Professor of Food Industry of Urmia University, Iran Assistant Professor of Microb Biotechnology, Institute of Biotechnology, Karaj, Iran Introduction: Ethanol made by a biomass is one of the useful strategies in terms of economic and environmental and as a clean and safe energy to replace fossil fuels considered and examined. Materials and methods: In this study, key enzyme in the production of ethanol (Pyruvate decarboxylase) from Zymomonas mobilis bacteria was isolated and cloned at E. coli bacteria by freeze and thaw method. For gene cloning, we used specific primers of pdc and PCR reaction and then pdc gene isolated and pET 28a plasmid double digested with (Sal I and Xho I) enzymes. Digestion Products were ligated by T4 DNA ligase in 16 °C for 16 hours. Results: Results of bacteria culture showed that a few colonies containing pET 28a plasmid could grow. Result of colony pcr of pdc gene with specific primers revealed 1700 bp bands in 1% agarose gel electrophoresis. The results of PCR with T7 promotor forward primer and pdc revers primer have proved the accurate direction of integration of pdc gene into plasmid and revealed 1885 bp band. Double digestion of recombinant plasmid with SalI and XhoI enzymes revealed same bands. Finally, RT showed the expected band of 1700 bp that implies the desired gene expression in the samples. Discussion and conclusion: Due to the increased production of ethanol via pyruvate decarboxylase gene cloning in expression plasmids with a strong promoter upstream of the cloning site can conclude that, pyruvate decarboxylase cloning as a key gene would be useful and according to beneficial properties of E. coli bacteria, transfering the gene to bacteria appears to be reasonable.http://uijs.ui.ac.ir/bjm/browse.php?a_code=A-10-545-1&slc_lang=en&sid=1Bioethanol fermentation gene transformation lignosellolusic material pdc gene pET 28a plasmid |
| spellingShingle | Masome Zeinali Bahman Hosseini Soodabeh Jahanbakhsh Mahmod Rezazadeh bari Meisam Tabatabaee Cloning of affecting pyruvate decarboxylase gene in the production bioethanol of agricultural waste in the E.coli bacteria Biological Journal of Microorganism Bioethanol fermentation gene transformation lignosellolusic material pdc gene pET 28a plasmid |
| title | Cloning of affecting pyruvate decarboxylase gene in the production bioethanol of agricultural waste in the E.coli bacteria |
| title_full | Cloning of affecting pyruvate decarboxylase gene in the production bioethanol of agricultural waste in the E.coli bacteria |
| title_fullStr | Cloning of affecting pyruvate decarboxylase gene in the production bioethanol of agricultural waste in the E.coli bacteria |
| title_full_unstemmed | Cloning of affecting pyruvate decarboxylase gene in the production bioethanol of agricultural waste in the E.coli bacteria |
| title_short | Cloning of affecting pyruvate decarboxylase gene in the production bioethanol of agricultural waste in the E.coli bacteria |
| title_sort | cloning of affecting pyruvate decarboxylase gene in the production bioethanol of agricultural waste in the e coli bacteria |
| topic | Bioethanol fermentation gene transformation lignosellolusic material pdc gene pET 28a plasmid |
| url | http://uijs.ui.ac.ir/bjm/browse.php?a_code=A-10-545-1&slc_lang=en&sid=1 |
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