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CRISPR is known to be present in approximately forty percent of bacteria and almost all in archea. It consists of 20-50 bases pairs generating RNA(CrRNAs) transcribed from a segment of foreign DNA. It uses this DNA to start the degradation of invading DNA that looks alike. Theses foreign DNA fragments called "spacers" are incorporated in CRISPR loci cas1cas2. It has been proven that spacers sequences are very variable and can be made from both coding and non coding regions of the foreign DNA. Although Cas 1 Cas2 are necessary for insertion of a protospacer"metal dependent nucleases", their functions during CRISPR Cas immunity remain unclear. The question is can Cas 1 cas 2 insert a protospacer in CRISPR loci in this particular case?
This work was made in order to understand the specificity of cas1cas2 binding in CRISPR spacer. One of the methods used in this research was gel filtration chromatography and size exclusion chromatography.These techniques were demonstrated in our very first lab session of Chem 378. The insertion of 33 bases pairs spacer derived from foreign spacer demonstrated that the nucleases Cas1Cas2 are the only proteins needed for CRISPR spacer acquisition. The gain insights into the structural organization of cas1cas2 complex using gel filtration and SAD, showed that the hydrophobic interactions between them triggered a conformational change and the amino acid Alanine at cas1 active site terminated and the other at cas2 mutated .Consequently, one can conclude that cas 2 can still acquire new spacers allowing a non enzymatic role of the cas2 during immunity.
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