Azza- Entry #4

One of my side projects was getting to make more of an enzyme called phusion that is used in PCR. Phusion is similar to the Taq polymerase were more familiar with, but the difference is that phusion is a lot more heat stable because it was first discovered in hot springs and therefore can withstand high annealing temperatures. It was definitely not easy trying to purify phusion with the FPLC, but once I was finally able to the amount of phusion I was able to replicate was worth up to $168,000! Taq is available for commercial purchase, but it’s also very expensive, so most labs (if they have the time) prefer to make more phusion on their own. The process is very straight-forward. You began with a phusion stock that is stored in the -80 degree. You use a pipette tip and scrap some phusion out and place it in a 25mL starter culture of broth (either LB or 2XYT). Because phusion has a kanamycin and chloramphenicol resistance gene, we used 25 uL of each to have a selection component. The next day we use the starter culture to induct a larger culture of 750 mL of bacterial broth. Then we spin down the larger culture and suspend the pellet in lysis buffer and sonicate and heat it up and finally run it on the FPLC. The FPLC is a machine basically composed of anion and cation exchange tubes that allow for the purification of phusion. To test if we’ve purified phusion we run the sample on a protein gel. The image below are my results after performing the protein gels. The different wells are different concentrations of phusion which were diluted with nuclease-free water. The last well is the commercial phusion we used to compare the one I made to. You can see how concentrated the phusion I made, which shows that they can now start using the phusion I made!