Final Purification?

Welcome back everyone! As you know from last week, I created a 1L batch of my pfu producing Bl21, sonicated it, and centrifuged it, but it still contained DNA leftover from the lysed E. Coli. If we were to use the solution I created for PCR in this state, it would work in replicating whatever DNA we want (if we put in the right primers and such, more on that next week), but it would also replicate the contaminating E. Coli DNA and separating DNA can be a pretty difficult and expensive process. Now, how do we get rid of this contaminating DNA? Well, its actually very simple. All we had to do was treat the pfu solution with an enzyme called DNase I. DNase I, when put in the proper buffer, will indiscriminately cleave DNA leaving it in very tiny fragments. These fragments are so small that they will not be replicated in PCR.

Now that we added the DNase and it has done its job, how do we get rid of it, as it will also cleave the DNA we want to clone for PCR? There is also a very easy solution to this too! Just put it in an 80°C water bath, and watch it denature! This part is actually kinda cool to watch because the pfu solution goes from a relatively clear color to a very cloudy mixture as the DNase and other contaminating proteins denature (I wish I had taken some pictures).

After one more centrifugation to remove the precipitated proteins and such, we are now ready for the final step before the pfu can really be used, dialysis. Dialysis is the process of removing things from a solution, think blood dialysis. For our dialysis we used a centrifuge and put the pfu solution in a special tube with a filter in it that lets all molecules smaller than 10kDa slip through. Pfu is 95kDa so we do not need to worry about losing it. Not only does this dialysis remove some unwanted molecules, it also can be used to change the buffer that contains the pfu, since it is not in the most stable buffer for storage. When we centrifuge the solution, we want to make sure that there is at least some of the original solution left, otherwise we will blowout all the pfu through the filter membrane. Then, after the centrifugation, we add the storage buffer, centrifuge, add storage buffer, centrifuge, and keep doing that until we think the original buffer has been diluted enough. Next, we add glycerol, which helps to further stabilize the solution so we can freeze it, to the solution until it makes up 50% of it, and finally we are done! Not quite though, we still need to test if it actually works. Stay tuned next, next week, as I will be on spring break, to see if the pfu actually works!

Increasing the Scale

Welcome back everyone! This week taught me about just how much waiting there can be for experiments. We decided that it was time to start increasing the amount of bacteria we were producing by ten fold, going from 100mL to 1L of bacteria. Much of my time was spent waiting for the bacteria to grow (about 2 hours after having grown it overnight), then waiting for the bacteria to induce (4 hours), and a bunch more waiting from processes like centrifugation or running a gel or waiting for something to heat/cool. There is just so much waiting! Although, all of this waiting did allow me to finally straighten up my notes and label my SDS-PAGEs on PowerPoint, which I had to learn how to use.

Was finally able to label this and make it look all pretty! All Sizes are given in kDa.

From some prior research that I had received from my on-site mentor Dr. Gustin, I found that the size of pfu is about 95kDa. But, the research that I had obtained that number from also used a different type of pfu; a type that has a histidine tag. Histidine is an amino acid that has an attraction to metals like Ni, nickel. This means that if a protein has a bunch of histidine attached to its end, it will stick to that metal, and thus purify itself because it will be the only protein sticking to the metal. The pfu that I am making does not have a tag, so we are planning on purifying it through simple heat treatment, aka making it so hot that all other proteins fall apart leaving pfu by itself as it is thermostable. As of right now, I have made a protein soup by sonicating the 1L culture of bacteria after centrifuging it down into a pellet and re-suspending it. We could use the pfu in PCR right now, but it is probably way too concentrated plus we do not know the proper dilutions, and it still has DNA from the E. Coli in it. Next week, I will remove the DNA by treating it with an enzyme called DNase and hopefully further purify my pfu. Thank you!