Eat, sleep, breathe, run chromatography columns

I would rate the past 72 hours as the most intense (and at times stressful) yet rewarding in my lab experience thus far. It all began last week when I was preparing some DnaK mutant protein to ship to Sigurd, who is currently traveling all over the U.S.--from his hometown in Oregon to Princeton to Stanford, where he will be performing Small Angle X-ray Scattering (SAXS) experiments. On Tuesday I finished dialyzing the protein, which Sigurd and I had purified together several weeks ago, and per his instructions packed it in a styrofoam box with water ice and poured liquid nitrogen on top. I then sealed the box with tape and brought it to the administers upstairs, where a man from TNT picked up the thankfully no longer smoking box and sent it on its way to Palo Alto. Sigurd sent me a letter to attach to the package explaining that the recombinant protein was non-pathogenic and purified from the harmless lab bacterium E. coli.

Three days later, on Friday, I received a call in lab from the secretary saying they needed to see me immediately regarding the protein shipment to Stanford. I was sure that the package had exploded due to evaporation of the liquid nitrogen I poured on top and headed to the secretary expecting the worst. It turned out that the package had not exploded but was being held at U.S. customs because the description of the protein was not adequate. I called and emailed Sigurd, who soon sent me a longer letter to send to TNT customer service in the States. The letter explained the fragility of the protein, that it was sent only on water ice, and that it had to be kept cold. I forwarded the letter to TNT and apparently Sigurd has still not heard about the status of the package. We can only hope that it is being chilled while the paperwork is completed.

In the meantime, Sigurd requested that I purify a variant of the original protein to give to his wife Erika in a thermos. Erika is flying out to join Sigurd this Wednesday, and we arranged for her to pick up the protein Tuesday afternoon. She said in an email that she couldn't wait to tell airport security that she was carrying a thermos containing biological materials from a man she barely knew.

And so my mission, if I chose to accept, was to purify a DnaK mutant beginning only with a plasmid encoding the protein, and have it ready by Tuesday afternoon. This meant transforming E. coli with the plasmid, growing up "heaps" of bacteria, lysing the cells, and then running three chromatography columns plus several rounds of dialysis with various buffers. I had done all of these things once before under the watchful eye of Sigurd (he also did some parts without me there while I took care of administrative issues the first couple weeks) and in twice the amount of time that I currently had.

As I began the challenge, I already had a draft of the email to Sigurd in my mind explaining the horrible mistake I had made and how I lost all the protein in some massive waste beaker or something. I was really not expecting to be successful--there were too many complex steps that I had observed only once or not at all. There was bound to be a major error at some point. And when the transformation and cell lysis went smoothly, I thought to myself 'Things are going too well, I'm going to screw up the chromatography.'

On Saturday morning, I got up at 7 AM and arrived in lab at 8 to spin down cells for lysis. I had them chilling on ice by 9 and and ran back to Carrington to meet up with the other summer students to go hiking. I had promised that I would take everyone up the Mt. Cargill trail and we would go to Capers for a pancake brunch afterward. When I told them that we actually wouldn't be back until 2 or 3, they protested that brunch is strictly between breakfast and lunch. Haven't they heard of Greylock brunch night? If you're eating pancakes, no matter the time, you're having brunch. The others were non-hikers but good sports who did well on the 5 hour round-trip journey (pictures soon). We returned to town to find Capers closed, causing temporary despair until we decided on hamburgers at Velvet Burger. When we finished at 4 pm, I walked back to lab to lyse cells and begin the first column, a DEAE anion exchange column. The main problem I ran into was setting up the plumbing connecting the pump to the column. Fishing through multiple boxes of metric and U.S. adapters and tubing, I got pretty creative in setting up a system that seemed to work with no leaks. At 11 pm I went into town for a quick dinner and shower, then back to lab until 5:30 am, equilibrating the column, loading the sample, collecting the appropriate fractions, running gels to see which fractions had the DnaK, cleaning the column, and then setting up the dialysis for "overnight" (i.e. while I slept).

I woke up at 2 pm on Sunday and headed back to lab to run column #2, the ATP affinity column. DnaK has high affinity for ATP, so this is the primary column in the purification. The main challenge this time was that the column kept going over the pressure limit and had to be run at a miniscule flow rate, I imagine because the column was old and needed to be re-set, thus dramatically augmenting the time required. By early evening I set up a program to load the sample and elute in fractions with the appropriate buffer and took off for the University gym for some much-needed exercise. A couple hours later I returned, completely prepared for A) the column to have exploded, B) the fraction collector to have malfunctioned, leaving my sample in a pool on the benchtop, C) the program to have pumped in the incorrect buffer, D) something worse. Miraculously, everything seemed to be working well and a gel confirmed which fractions contained DnaK. I was in bed early at 3 AM.

I arrived today back in lab by noon and was happy to see that the program I had run while asleep to equilibrate column #3, gel filtration, had worked swimmingly. I injected the sample onto the gel filtration column and watched the computer as a beautiful UV absorbance peak emerged right where it was expected. I just finished running gels on fractions jam packed with DnaK.

All that is left is two more dialysis runs and some flash freezing and she'll be ready for Erika. The weekend taught me A) working in the lab alone to Young Gs by P. Diddy at 4 am is quite enjoyable, B) coffee actually tastes pretty good, C) don't doubt myself so much.