Développement d'un construct de gène rapporteur pour la localisation subcellulaire
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La pyrophosphohydrolase d'ARN (RppH) d�clenche la d�gradation des transcrits en �liminant le pyrophosphate de l'extr�mit� 5' des ARNm, ce qui permet la liaison de la RNase E/RNase J chez les bact�ries. Une ARN pyrophosphorylase putative est pr�sente dans les cellules de C. reinhardtii et plusieurs indices sugg�rent que cette prot�ine est impliqu�e dans la d�gradation de l'ARNm au sein du chloroplaste. Afin de d�terminer la localisation de la prot�ine dans les cellules de Chlamydomonas, l'extr�mit� 5' du g�ne rppH a �t� marqu�e par une prot�ine fluorescente verte � codons optimis�s et introduite dans des cellules de Chlamydomonas. La GFP (Zsgreen 1) a �t� optimis�e au niveau des codons � l'aide d'une base de donn�es sur l'utilisation des codons. Le cadre de lecture de la GFP synth�tique optimis�e a �t� ajust� et clon� dans le vecteur pBluescript-5'rppH SK+. Le fragment de g�ne 5'rppH-GFP a ensuite �t� clon� dans trois vecteurs. Toutes les constructions ont �t� v�rifi�es. Les transformants ont �t� cribl�s par PCR pour d�tecter la pr�sence du g�ne GFP chim�rique. Les exp�riences de criblage nous ont amen�s � choisir un transformant pouvant �tre utilis� pour la suite des travaux. De plus, bien que la fluorescence n'ait pas �t� v�rifi�e, la GFP synth�tique optimis�e a �t� exprim�e dans des cellules E. coli (BL21DE3), confirmant la fonctionnalit� de la construction GFP synth�tique in vivo.
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Rich Dad Poor Dad: What the Rich Teach Their Kids about Money That the Poor and Middle Class Do Not!
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