What I'm trying to do - layman version
RtcB is an E.coli protein ligase - meaning, an enyzme that attaches two pieces of RNA together. I'm trying to purify it so that I can attach it to a 13 nanometer (nm) gold nanoparticle later. We're using a protein rather than a DNAzyme (~20 nucleotide DNA that acts like an enzyme) because the rate of the ligation (attaching) reaction was too slow.
Experiments This Week
Monday – prepared “buffer A,” inoculated culture
Tuesday – protein expression of rtcB
Wednesday – cleaning and reading day
Thursday – protein expression of rtcB
Friday – protein purification of rtcB via sonication and Ni-NTA affinity column
Eppendorf centrifuge 5804R (Lynn lab) – We also have one in the cell culture room.
Optima XE-90 Ultracentrifuge (Weinert lab)
I performed protein purifications in the Lynn lab. The post-doc Dan showed me where everything was and the basics of the operation of the incubator/shaker and autoclave. Rolando showed me how to use the spectrophometer. I was inefficient and made a lot of silly mistakes on Tu, including spilling 200 mls of culture while trying to balance for the 0.5 volume centrifuge in the Weinert lab. Cleaned everything with bleach liberally. Decided it would be best to repeat the experiment on Th.
Things went much better on Th. Centrifuged down my samples in 50 ml falcon tubes six and four at a time for 20 min 11,000 rpm on centrifuge listed under “equipment.” Kept them all on ice the entire time and froze them in -80C afterward (Lynn lab, room E439).
Took 1 ml samples of my culture every hour before and after protein expression with IPTG. Spun down each individually (not in a cooled centrifuged), dumped super and stored in -20C in Salaita lab.
On Friday, Yoshie showed Yuan and I how to purify the protein via sonication (Lutz lab) and purification via Ni-NTA column.
Purchases for the week = $0
What I Still Need to Do
1. Run an SDS-PAGE gel of protein fractions and final protein concentrate to determine purity and confirm it’s MW.
2. Determine why the 10mer oligo was not visible on the SDS-PAGE. I’ll be running a 20% gel rather than the 18% in the paper, to see if I can get better separation. Kevin was thinking of optimizing the system by trying to run gels of just the starting material oligos until we can see them. He used the oligold dye.
3. Look up the use of the different dyes for SDS-PAGE.
4. Look up info about synthesizing AuNPs and how long it takes.
5. Look up info on non-specific adsorption of protein to an AuNP.
6. Re-read Church and Zhang papers on Cas9.
7. Look up more info on Cas9 mechanism.
8. Do a lit search on AuNP toxicity studies.
9. Do a lit search on thiol bond (lipoic acid) binding affinity studies (search drug release).
10. Read the Cas9 papers and prepare for a literature talk in group meeting to discuss them and pitch my idea.
11. Make more plates at some point, to preserve the transformation. The amp/km plates I got from the Lutz lab are way too skinny.
Tentative Schedule for Next Week
Start examining how to clone GBP into pQE70 and if I can figure it out, do that in parallel with the other experiments. Should involve… 1) synthesis or purchase of GBP 2) digestion of pQE70 2) ligation 3) transformation 4) isolation 5) confirm the sequence 6) transformation, expression, purification of protein
Monday – prepare 12% SDS-PAGE, run protein gel control and one to check protein purity. Prepare 20% SDS-PAGE gel for oligos; run starting materials
Tuesday – If oligo gel works, prepare and perform ligation reaction
Wednesday – Run ligation reaction on a gel
Thursday – TBA
Friday – TBA
Make sure to have elution buffer ready before you do your Ni-NTA column. Yes, go ahead and filter 100 ml of the elution buffer. Keep all buffers in cold room at all times.
OD600 measurements are dimensionless units like klett and vary depending on the instrument, bulb and filter. Note the filter that’s in the Lynn lab spec. To relate them to cells/ml, will need to do cell counting experiments with several dilutions.
Remember to turn on the spec 30 min before you need to use it to allow the bulb to warm.
The Lynn lab incubator in room E439 shuts off the shaker part when the temperature is changed. Must turn it back on manually each time you change the temperature.
Grow 20-30 ml o/ns when doing expression in 0.5 L volumes. Make sure o/n don’t grow longer than 12-16 hrs.
Autoclave the 0.5 L of LB in the flask that you will grow your culture in to avoid having to transfer it.
Make sure to prepare your microcentrifuge tubes ahead of time with labels and the falcon tubes that you’ll store in -80C.
Sonicator volume limit in the Lutz lab is 10 ml, so do not sonicate more than that. Do 10 sec pulses, rest 20 sec and repeat for 4 min.
Do not violently shake the Ni-NTA agarose or you could degrade the beads. Gently swish back and forth to resuspend.
Use 1 ml of 50% Ni-NTA slurry for every 4 ml of cleared lysate. More than 3 ml is probably plenty, regardless of the lysate.
Do not let the Ni-NTA slurry/lysate incubate longer than one hour, to avoid potential damage to the protein via proteases (whatever hasn’t been inactivated by the cocktail).
Amicon ultra centrifugal filter columns are located in the drawer right next to the main door on the right.
You can flash freeze store your protein in nitrogen and keep in -80C. Should keep for at least a year.