Optimisation of transfor mation method of E ragrostis tef and Eragrostis curvula orphan crops / Ewa Bachul

Public ISBD Titre : Optimisation of transfor mation method of E ragrostis tef and Eragrostis curvula orphan crops Type de document : TFE / Mémoire Auteurs : Ewa Bachul, Auteur ; Jonathan Scauflaire, Directeur de la recherche Editeur : Montignies-sur-Sambre : Helha. Agronomie Année de publication : 2022 Note générale : Le fichier numérique de ce document est disponible uniquement pour les membres de la Haute Ecole Louvain-en-Hainaut ainsi que ses étudiants. Veuillez-vous connecter pour accéder à votre compte lecteur Langues : Français (fre) Index. décimale : TFE AIBT TFE Agro-industries et biotechnologies Résumé : This work reports on experiments conducted at the University of Cape Town in South Africa with the aim of optimizing the genetic transformation of Eragrostis tef and Eragrostis curvula, two orphan plants from Africa. The overall aim was to insert foreign DNA into these plants while being able to verify the effectiveness of the transformation. The work was divided into several steps and involved the most common molecular biology techniques such as PCR, DNA extraction, etc. The experiments began with the preparation of the cloning vector. mCherry, a chromophore allowing post-experimental verification, was inserted into pB2WG7, a plasmid. In the second step, it is necessary to verify that the insertion has been done correctly before the cloning vector can be placed in culture with Escherichia coli. After that, the bacteria themselves will be placed in Agrobacterium tumefaciens. The latter will infect the plants so that they can integrate this new genetic material into their genome. The last step, verification, is possible thanks to mCherry. The aim of spectrophotometry is to see if it is visible at the wavelength to which it is supposed to fluorescence and conclude that the experiments are working correctly. The results can be considered promising and worthwhile, but unfortunately, science is an unpredictable field and has not always allowed the experiments to achieve in their entirety. Note de contenu : 5 Table of contents Acknowledgements............................................................................................................ 4 .......................................................................................................................................... 6 I. Introduction to the University ..................................................................................... 7 II. General content.......................................................................................................... 8 1. Introduction ........................................................................................................................ 8 2. Theoretical concepts ........................................................................................................... 9 2.1. Eragrostis tef ................................................................................................................................... 9 2.2. Eragrostis curvula ............................................................................................................................ 9 2.3. pEAQ HT......................................................................................................................................... 10 2.4. mCherry ......................................................................................................................................... 10 2.5. pB2WG7......................................................................................................................................... 10 2.6. PCR................................................................................................................................................. 11 2.7. Hi-Fi PCR ........................................................................................................................................ 11 2.8. DNA electrophoresis ...................................................................................................................... 12 2.9. Quantification of nucleic acids ...................................................................................................... 12 2.10. Agrobacterium tumefaciens .......................................................................................................... 13 2.11. Screening ....................................................................................................................................... 14 2.12. Escherichia coli .............................................................................................................................. 15 III. Objectives and strategy .........................................................................................16 IV. Materials and methods ..........................................................................................18 1. Routine procedures ........................................................................................................... 18 1.1. Preparation of culture media ........................................................................................................ 18 1.2. Sterilization methods..................................................................................................................... 19 1.2.1. Sterilization of culture media.................................................................................................... 19 1.2.2. Sterilization of diluted solutions ............................................................................................... 19 1.3. Sterilization of Eragrostis tef and Eragrostis curvula seeds........................................................... 19 1.4. Seed culturing ................................................................................................................................ 20 1.5. Preparation of the plasmid ............................................................................................................ 20 1.6. Plasmid DNA extraction ................................................................................................................. 20 1.7. Concentration measurement ........................................................................................................ 21 2. Experiments performed .................................................................................................... 21 2.1. Plasmid digests .............................................................................................................................. 21 2.1.1. Amount of DNA ......................................................................................................................... 21 2.1.2. Digestion of pEAQ HT................................................................................................................ 21 2.1.3. Digestion of pB2WG7................................................................................................................ 22 2.2. mCherry amplification by PCR ....................................................................................................... 22 2.2.1. PCR mCherry to ligate with pEAQ HT ........................................................................................ 22 2.2.2. PCR of mCherry to ligate with pB2WG7 ................................................................................... 23 2.3. Digestion of mCherry ..................................................................................................................... 23 2.4. Ligation .......................................................................................................................................... 23 2.5. Preparation of competent cells ..................................................................................................... 24 2.6. Bacterial transformation ............................................................................................................... 24 2.7. Colony PCR..................................................................................................................................... 24 2.8. Plasmid isolation............................................................................................................................ 25 2.9. Verification of the ligation between pB2WG7 and mCherry ......................................................... 25 2.9.1. PCR validation ........................................................................................................................... 25 2.9.2. Antibiotic validation .................................................................................................................. 25 2.9.3. Check 3 by digestion ................................................................................................................. 25 2.9.4. PCR validation 2 ........................................................................................................................ 26 6 2.10. Preparation and infection of Agrobacterium ................................................................................ 26 2.10.1. Making Agrobacterium electrocompetent .......................................................................... 26 2.10.2. Transformation of Agrobacterium by electroporation ........................................................ 27 2.10.3. Verification of recombinant Agrobacterium ........................................................................ 27 2.10.4. Agrobacterium induction ..................................................................................................... 27 2.10.5. Agrobacterium infiltration ................................................................................................... 27 2.11. Infection test on callus of curvula.................................................................................................. 28 2.12. Check for leaves infection by proteins extraction and Bradford assay ......................................... 28 2.13. Check for leaves infection by SDS PAGE ........................................................................................ 29 V. Results ......................................................................................................................30 1. Plasmids digests ................................................................................................................ 30 2. mCherry amplification by PCR ........................................................................................... 30 3. Verification of the ligation between pB2WG7 and mCherry .............................................. 31 4. Check for leaves infection by proteins extraction and Bradford assay................................ 33 5. Check for leaves infection by SDS PAGE ............................................................................. 33 VI. Interpretations ......................................................................................................34 VII. Conclusion .............................................................................................................37 VIII. Glossary.................................................................................................................38 IX. Table of figures ......................................................................................................41 X. Tables list ..................................................................................................................42 XI. Abbreviations ........................................................................................................43 XII. Bibliography ..........................................................................................................44 Permalink : ./index.php?lvl=notice_display&id=105987 Optimisation of transfor mation method of E ragrostis tef and Eragrostis curvula orphan crops [TFE / Mémoire] / Ewa Bachul, Auteur ; Jonathan Scauflaire, Directeur de la recherche . - Montignies-sur-Sambre : Helha. Agronomie, 2022. Le fichier numérique de ce document est disponible uniquement pour les membres de la Haute Ecole Louvain-en-Hainaut ainsi que ses étudiants. Veuillez-vous connecter pour accéder à votre compte lecteur Langues : Français (fre) Index. décimale : TFE AIBT TFE Agro-industries et biotechnologies Résumé : This work reports on experiments conducted at the University of Cape Town in South Africa with the aim of optimizing the genetic transformation of Eragrostis tef and Eragrostis curvula, two orphan plants from Africa. The overall aim was to insert foreign DNA into these plants while being able to verify the effectiveness of the transformation. The work was divided into several steps and involved the most common molecular biology techniques such as PCR, DNA extraction, etc. The experiments began with the preparation of the cloning vector. mCherry, a chromophore allowing post-experimental verification, was inserted into pB2WG7, a plasmid. In the second step, it is necessary to verify that the insertion has been done correctly before the cloning vector can be placed in culture with Escherichia coli. After that, the bacteria themselves will be placed in Agrobacterium tumefaciens. The latter will infect the plants so that they can integrate this new genetic material into their genome. The last step, verification, is possible thanks to mCherry. The aim of spectrophotometry is to see if it is visible at the wavelength to which it is supposed to fluorescence and conclude that the experiments are working correctly. The results can be considered promising and worthwhile, but unfortunately, science is an unpredictable field and has not always allowed the experiments to achieve in their entirety. Note de contenu : 5 Table of contents Acknowledgements............................................................................................................ 4 .......................................................................................................................................... 6 I. Introduction to the University ..................................................................................... 7 II. General content.......................................................................................................... 8 1. Introduction ........................................................................................................................ 8 2. Theoretical concepts ........................................................................................................... 9 2.1. Eragrostis tef ................................................................................................................................... 9 2.2. Eragrostis curvula ............................................................................................................................ 9 2.3. pEAQ HT......................................................................................................................................... 10 2.4. mCherry ......................................................................................................................................... 10 2.5. pB2WG7......................................................................................................................................... 10 2.6. PCR................................................................................................................................................. 11 2.7. Hi-Fi PCR ........................................................................................................................................ 11 2.8. DNA electrophoresis ...................................................................................................................... 12 2.9. Quantification of nucleic acids ...................................................................................................... 12 2.10. Agrobacterium tumefaciens .......................................................................................................... 13 2.11. Screening ....................................................................................................................................... 14 2.12. Escherichia coli .............................................................................................................................. 15 III. Objectives and strategy .........................................................................................16 IV. Materials and methods ..........................................................................................18 1. Routine procedures ........................................................................................................... 18 1.1. Preparation of culture media ........................................................................................................ 18 1.2. Sterilization methods..................................................................................................................... 19 1.2.1. Sterilization of culture media.................................................................................................... 19 1.2.2. Sterilization of diluted solutions ............................................................................................... 19 1.3. Sterilization of Eragrostis tef and Eragrostis curvula seeds........................................................... 19 1.4. Seed culturing ................................................................................................................................ 20 1.5. Preparation of the plasmid ............................................................................................................ 20 1.6. Plasmid DNA extraction ................................................................................................................. 20 1.7. Concentration measurement ........................................................................................................ 21 2. Experiments performed .................................................................................................... 21 2.1. Plasmid digests .............................................................................................................................. 21 2.1.1. Amount of DNA ......................................................................................................................... 21 2.1.2. Digestion of pEAQ HT................................................................................................................ 21 2.1.3. Digestion of pB2WG7................................................................................................................ 22 2.2. mCherry amplification by PCR ....................................................................................................... 22 2.2.1. PCR mCherry to ligate with pEAQ HT ........................................................................................ 22 2.2.2. PCR of mCherry to ligate with pB2WG7 ................................................................................... 23 2.3. Digestion of mCherry ..................................................................................................................... 23 2.4. Ligation .......................................................................................................................................... 23 2.5. Preparation of competent cells ..................................................................................................... 24 2.6. Bacterial transformation ............................................................................................................... 24 2.7. Colony PCR..................................................................................................................................... 24 2.8. Plasmid isolation............................................................................................................................ 25 2.9. Verification of the ligation between pB2WG7 and mCherry ......................................................... 25 2.9.1. PCR validation ........................................................................................................................... 25 2.9.2. Antibiotic validation .................................................................................................................. 25 2.9.3. Check 3 by digestion ................................................................................................................. 25 2.9.4. PCR validation 2 ........................................................................................................................ 26 6 2.10. Preparation and infection of Agrobacterium ................................................................................ 26 2.10.1. Making Agrobacterium electrocompetent .......................................................................... 26 2.10.2. Transformation of Agrobacterium by electroporation ........................................................ 27 2.10.3. Verification of recombinant Agrobacterium ........................................................................ 27 2.10.4. Agrobacterium induction ..................................................................................................... 27 2.10.5. Agrobacterium infiltration ................................................................................................... 27 2.11. Infection test on callus of curvula.................................................................................................. 28 2.12. Check for leaves infection by proteins extraction and Bradford assay ......................................... 28 2.13. Check for leaves infection by SDS PAGE ........................................................................................ 29 V. Results ......................................................................................................................30 1. Plasmids digests ................................................................................................................ 30 2. mCherry amplification by PCR ........................................................................................... 30 3. Verification of the ligation between pB2WG7 and mCherry .............................................. 31 4. Check for leaves infection by proteins extraction and Bradford assay................................ 33 5. Check for leaves infection by SDS PAGE ............................................................................. 33 VI. Interpretations ......................................................................................................34 VII. Conclusion .............................................................................................................37 VIII. Glossary.................................................................................................................38 IX. Table of figures ......................................................................................................41 X. Tables list ..................................................................................................................42 XI. Abbreviations ........................................................................................................43 XII. Bibliography ..........................................................................................................44 Permalink : ./index.php?lvl=notice_display&id=105987

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