Azza- Entry #3

Since I've come into the Lab, I was given my own personal project to work on. The goal of this project is to clone a plasmid responsible for creating Cas9. Cas9 is a protein that is a part of the CRISPR/Cas9 method of cleaving out around 20b.p of DNA and inserting foreign DNA into a cell line or an organism in order to observe a mutation of a targeted gene. Cas9 is an RNA guided DNA endonuclease enzyme. To clone CAs9 we are using a plasmid created by Martin Jinek, who once worked for my PI here at UCSF. Martin Jinek was also the student working for Jennifer Doudna when she discovered CRISPR/Cas9. Jinek is also the first author on some very famous papers that introduced the world to CRISPR/Cas9. The way I started off with this project was to convert the word document I've received from Martin Jinek for the coding of the MJ922 plasmind to an application called ape which lets you annotate certain regions of the plasmid. Then we got the plasmid from a postdoc student in the lab and transformed it using Rosetta cells. Rosetta cells are probably the most versatile cells used for transfection and transformation. Once we transformed the cells, we grew them at large scale. Then we spun them down and created large cultures and then extracted the  protein from the pellet using the FPLC machine. I only got so far with this project as time was running out and I wanted to stick with a different project of cloning and purifying more Phusion. 

this is what the annotation of the plasmid looked it, just to familiarize myself with what was being transformed.