, Avec cette souche, plusieurs colonies ont été obtenues sur les boites SP4 tétracycline 5 µg/mL, et ont été repiquées en milieu liquide. A nouveau, un seul clone s'est multiplié en milieu sélectif. L'ADN génomique de ce clone a été extrait, mpn110 et mpn347 ont été supprimés

, Le pad400 a été introduit dans la souche M. pneumoniae M129 ?mpn133 ?mpn491

, ?mpn110 ?mpn347 -pad372, précédemment obtenue en utilisant un protocole identique

, Conclusion Deux nouvelles souches de M. pneumoniae ont été obtenues. Elles contiennent un ou deux landing pads en fonction de la modification souhaitée (insertion ou remplacement). Ces souches sont donc prêtes à « accueillir » ou échanger une nouvelle portion de génome, Plusieurs colonies ont été obtenues sur les boites SP4 chloramphénicol 50 µg/mL, puis repiquées en milieu liquide. L'ADNg de 20 clones a été extrait

, En fonction de la modification envisagée, les différents vecteurs navettes ont été transformés par électroporation dans différentes souches « landing pad » de M

, Le plasmide pCC1YTRP-gmR-s10-cre a été transformé dans la souche M

. M129-pad014, De façon surprenante, après une semaine seulement d'incubation à 37°C, des centaines colonies sont apparues sur les boites SP4 gentamycine 100 µg/mL. Nous avons analysé en PCR multiplex l'ADN génomique de 20 colonies (Figure 85), Cette PCR multiplex a -265

, de méthyler le génome donneur avec cette MTase avant toute expérience de transplantation, afin que celui-ci soit protégé d'une éventuelle dégradation par la cellule receveuse ou bien si

, Lors de la transplantation de génomes, le génome donneur est « injecté » dans la cellule receveuse. A l'heure actuelle, nous n'avons pas de réelle connaissance sur les mécanismes mis en place au niveau cellulaire, entre le moment où le génome donneur entre dans la cellule receveuse et l'obtention de la cellule mutée

. Lartigue, Du fait de l'absence de machinerie de recombinaison chez M. pneumoniae, cela ne devrait pas impacter la transplantation de génome dans notre cas. De plus, ce phénomène de recombinaison entre génomes n'a pas encore été démontré, 2007.

. Krypuy, Pour cela, nous pouvons prévoir un moyen de différencier les transplants attendus des transplants recombinants, le cas des transplantations interespèces, ou intra-espèces mais entre des souches différentes de M. pneumoniae, il pourrait être intéressant de mettre en place un test de PCR quantitative « High Resolution Melt » (qPCR-HRM), 2007.

, Il s'agit d'une PCR basée sur l'analyse des courbes de fusion de deux brins

X. , En revanche, ce test ne pourra pas être utilisé dans le cadre d'une transplantation de la souche M129 dans la souche M129, ou de la souche FH dans la souche FH. Dans ce cas-là, nous pourrions faire séquencer le génome du transplant afin de vérifier qu'il correspondrait bien à ce qui serait attendu. Bien entendu, nous ne pourrions pas, ici, déterminer si le mutant obtenu est un véritable « transplant » ou bien s'il s'agit d'un simple « recombinant », mais si au final le génome contient les modifications souhaitées, cela a peu d'importance. 3. 4. Le cytosquelette Un dernier élément que nous avons suspecté comme pouvant être une barrière à la transplantation de génomes est la présence d'un cytosquelette, mycoides, les souches PG1 et T1/44. Nous pourrions donc certainement l'adapter à M. pneu--266 --moniae, par exemple pour différencier les souches M129 et FH entre lesquelles il pourrait y avoir jusqu'à 1500 SNPs, 2006.

. García-morales, Celuici, localisé au niveau de la face interne de la membrane plasmique, est organisé sous la forme d'un réseau hélicoïdal. Il est composé d'éléments protéiques formant un assemblage régulier de mailles et entourant l'ensemble du cytoplasme. De nombreux éléments structurels, sous forme de « broches », sont présents de manière régulièrement espacée et relient le cytosquelette à la membrane cytoplasmique (Mayer, 2006), 2006.

B. Bose and . Krause-;-pich, Il s'agit d'une structure en forme de pointe, qui se situe à l'extrémité avant de la cellule, 2008.

, Nous avons donc émis l'hypothèse qu'une sont en cours d'obtention au laboratoire. Plus précisé--267 --ment, il s'agit de mutants M. pneumoniae ciblés pour les gènes mpn141 et mpn310, codant pour l'adhésine P1 et la protéine HMW2, respectivement ; et des mutants M. genitalium ciblés pour les gènes mg218, mg191 et mg192, codant pour un homologue de la protéine HMW2, l'adhésine P140, Le cytosquelette pourrait donc constituer une barrière physique à l'entrée du génome donneur dans la cellule receveuse (Figure 93 D)

, De plus, il a été observé, lors de l'obtention de mutants de délétion pour le gène mg218

M. Pich, Cette caractéristique pourrait elle aussi conférer un avantage positif à la transplantation, en permettant d'agrandir l'espace disponible pour l, 2008.

, Ces travaux de thèse ont porté sur le développement d'outils de biologie de synthèse permettant la construction d'une cellule minimale et non pathogène, à partir de M. pneumoniae. La stratégie adoptée a été d'utiliser la levure S. cerevisiae comme plateforme d'ingénierie de génomes, de nombreux outils de modification génétiques existant pour cet hôte

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