Week 6 was about taking all the DNA work from the previous weeks and finally turning it into something functional. The main focus was continuing to work with the pMIG-II plasmid and understanding why it is the plasmid being used. pMIG is useful because it has two very important features. One is GFP, which makes cells glow green if the plasmid is inside them. The other is ampicillin resistance, which allows bacteria carrying the plasmid to survive when grown on plates with antibiotics. If bacteria grow and glow, it means the cloning steps worked.
Above is what GFP would look like under a fluorescent microscope.
After running more restriction enzyme digestions, the pMIG-II backbone was separated using gel electrophoresis. From the gel, the correct DNA band was carefully cut out and purified using gel extraction. Gel extraction is a cleanup step that isolates only the DNA piece needed and removes agarose, enzymes, and extra fragments. This is important because dirty or mixed DNA can interfere with later steps. By the end of this step, the plasmid backbone was clean and ready to be joined with the insert.
The next new technique was ligation, which is the step where the insert DNA is attached to the plasmid backbone. Both the pMIG backbone and the FLT3-ITD insert had matching sticky ends because they were cut with the same restriction enzymes. A ligase enzyme was added to connect these ends and seal the DNA together. A 10:1 ratio of insert to vector was used, meaning there was much more insert than plasmid, which increases the chance that the insert goes into the plasmid instead of the plasmid reconnecting by itself. This reaction was set up in a total volume of 20 microliters and left to run so the DNA pieces could fully join.
The image on the right is actually an agar plate with some of my results on it. As you can see, the little white dots are the bacteria that have grown on the plate. The process of getting the products from our ligation reaction onto this agar plate is called bacterial transformation. This process moves the newly made plasmid into bacteria so it can be copied many times. The bacteria were mixed with the ligation product and then placed in a shaker for about one hour. This recovery time allows the bacteria to adjust and start expressing the genes on the plasmid. After that, the bacteria were plated onto agar plates that contained ampicillin. Because pMIG has ampicillin resistance, only bacteria that successfully took in the plasmid should be able to grow on these plates.
Everything is coming together but time is running out. I hope we can get the results we want before I have to start school again. Time is of the essence.
