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Hello!

Welcome to the Lab Notebook Wiki for Tube Tech! We're a team based out of Hess Laboratory at Columbia University's Biomedical Engineering Department.

This summer, we designed a protocol that produces EXCEPTIONALLY LONG MICROTUBULES! For details on our team, please check out the link below to our Wiki page:

Tube Tech!

Summary for May and June

During May and June research was done on the structure of microtubules. We learned the basic motility assay protocol as well as the chemical dynamics of microtubule polymerization. We learned how to use the microscope and employ fluorophores and fluorescent excitation in imaging through the Zyla and DU8157 cameras.

Logs for July

For the first weeks of July, preparations were made for the experiments to be carried out in the next few weeks. This involved finalizing experimental plans, reviewing information and ensuring that the reagents required were in stock.

7-20
polymerized tubulin and made 100x dilulted microtubule solution (MT100).
sheared the mt 100 solution using Hess/Jeune-Smith protocol. 250 µL of mt100 was passed through a 25 gauge needle with 1 mL syringe 5 times.
we imaged both sheared and unsheared microtubules under the microscope. There was no considerable difference between sheared and unsheared microtubule lengths.

7-21
microtubules sheared 7-20 were sheared again. They appeared fragmented when imaged.
these sheared microtubules were placed into a 37℃ water bath for 90 minutes.
both sheared and unsheared microtubules were diluted by 5 and the squashes were imaged.
We also imaged microtubules left to anneal in the water bath. These microtubules did appear to grow.
Seeding experiment
we took unsheared mt100 and sheared them using the same procedure. Polymerized 2 aliquots of tubulin with the seeds. Aliquot 1
12.5 µL mT100(sheared seeds), 1 µL GTP, 1 µL Mg, 1.2 DMSO, 9.3 µL BRB80. Aliquot2
12.5 µL MT100(sheared seeds), 1 µL GTP, 1 µL Mg, and 10.5 µL BRB80. Both aliquots were left to polymerize for 90 min.
After analyzing the data the unsheared microtubules had an average length of 13µm, sheared twice 6 µm, seeded without dmso 9 µm, seeded with dmso 17 µm.

7-25
We attempted to replicate the seeding experiment protocol outlined in the Mitchison and Kirschner paper on dynamic instability of microtubule growth(1984) with the hope that we may be able to repeat their results and increase average microtubule length to 30-40 µm.
Seeding Experiment #2
1) we polymerized tubulin using standard polimerization buffer, 8 µL for 1 aliquot of tubulin(final tubulin concentration of 25µM) at 37℃ for 30 min.
2) We then imaged a squash of the mt100 to see if the tubules had grown.
3) We used another aliquot of tubulin, this time with 13.33 µL polymerization buffer and no DMSO(just Mg,and GTP in BRB80), added buffer then incubated in ice for 5 min, then added 1.33 µL of seeds from the 25 µL polymerized earlier.(final tubulin 15 µM in solution), then set in 37℃ water bath for 30 min.
After analyzing the images using Fiesta the average mt100 lengths before seeding were 11.56 µm and 10.54 µm(taken from 2 images of the same solution). The mt100 lengths after seeding were 9.097 µm and 9.326 µm. In trying to replicate the results of this experiment, our resulting microtubules were on average 1 µm shorter. We were expecting to achieve a growth of up to 30-40 µm.
In the original protocol from the Kirshner paper glycerol was added to the polymerization buffer. Adding glycerol may yield desired growth.

7-27
Repeated seeding experiment from 7-25 with a few changes. We used a mixture of hylite and rhodamine labeled tubulin. Microtubules seeds were made from hylite tubulin and added to rhodamine tubulin. We suspected that if the rhodamine grows using the hylite seeds, we will be able to directly measure its effectiveness by using different laser frequencies from the microscope.
seeds were produced at 32 µM tubulin (6.25 Growth Buffer) for first polymerization, diluted to 15 µm for second polymerization. Allowed first polymerization to run at 37℃ for 30 min, second for 1 hour
squash of hylite seeds showed long MTs.
squash of hylite seeds and Rhodamine tubulin post
seeding showed only rhodamine microtubules
no hylite seeds visible under 647 Tirf
Hylite seeds may break down naturally over time, also possible that immersing in ice caused breakdown

7-28
repeated same seeding procedure mentioned 7/27/16 with a few changes
1. In order to more closely replicate the Mitchison paper, we pre-warmed the tubulin before adding the seeds
2. This time the seeds were made with rhodamine tubulin and were added to hylite tubulin. Rhodamine tubulin was polymerized in 6.25 µL growth buffer for 30 min. After polymerization 6 µL of taxol was added. 1.33 µL rhodamine seeds were added to 13.33 µL growth buffer and hylite tubulin polymerized at 37℃ for 30 min
when we viewed our seeds, we noticed that they were mostly broken down and shorter than before.(It is possible that hylite polymerizes better)
We could not observe rhodamine tubulin attached to hylite tubulin
In order to more closely recreate the original paper we will add glycerol to the growing seeds


Logs for August

Experiments continued from July. The main body of the seeding experiments took place during this month.

8-1 Glycerol seeding protocol:
1 aliquot rhodamine tubulin
6.25 µL glycerol growth buffer
14 µL BRB80 100%glycerol
2.5 µL mg (10mM final)
1 µL GTP (1 mM final)
polymerized for 45 min at 37℃
imaging mt100 showed concentrated very short microtubules or free tubulin.
we added 3 µL of 40 µM tubulin to original polymerization solution. The 3 µL contained standard growth buffer with no DMSO. final tubulin was 15 µM in 8 µL. Polymerized for 1 hour at 37℃.
imaged again, this time the microtubules were much longer.
Diluted 0.5 µL of these seeds into 15 µM(in 5 µL) tubulin with standard growth buffer(without dmso), polymerized for 45 min at 37℃.
After imaging, seeds diluted in 15 µM did not seem to grow longer.
imaged non
diluted and diluted seeds after another hour. Diluted seeds looked much longer. Non
diluted hadn’t grown.

8-2
Split tubulin aliquot in 2. Half for making seeds, half to add later to the seeds.
added 3.5 µL BRB80 to aliquot
removed 1.75 µL to separate tube
Made Growth Buffer with:
7.5 µL glycerol
2.5 µL Mg
1 µL GTP
and added 1.375 µL to the tubulin aliquot. This was vortexed heavily, then polymerized for 30 min at 37℃. We began polymerizing at 11:26AM
2nd growth buffer:
10.56 µL BRB80
2.5 µL Mg
1 µL GTP
Began polymerizing diluted seeds at 1:30 pm
we added 0.8 µL seeds to 4 µL of 25 mM tubulin
Incubated diluted seeds for 3 more hours and imaged them around 4:30
We also imaged MT100 from diluted seeds made yesterday
We added 3 µL of BRB80 and 3 µL taxol (warmed to room temp) to diluted seeds, also kept MT100 from diluted seeds.

8-3
Imaged diluted seeds and seeds from 8-1 and 8-2
Seeds from 8-2 that had taxol directly added to polymerization mix broke down.
seeds from 8-1 grew longer
possibly longer than diluted

8-4
Imaged seeds and diluted seeds from 8-1 and 8-2
tried to repolymerize seeds from 8-1 (MT100):
5 µL growth buffer in 1 tubulin aliqout (standard gb w/o dmso, 2.5 µL Mg) added All 5 µL to 8 µL of MT100 seeds from 8-1 (not diluted seeds) to make 13 µL with 15 µM free tubulin, incubated at 37℃ for 1 hour
imaged after 1 hr polymerization
there was nucleation but seeds didn’t grow longer (imaged 100x dilution and taxol)
Left the concentrated seeds and tubulin 15 µM in the incubator at 37℃ overnight
Also left the 100x dilution in the incubator overnight

8-5
Repolymerization/continuous polymerization:
began with 1 aliquot of tubulin split into 2 3.5 µL BRB80, 1.75 µL kept for polymerization and the other stored in ice for later.

Growth Buffer with 7.5 µL glycerol,2.5 µL Mg, 1 µL GTP Added 1.375 µL Growth Buffer to the 1.75 µL tubulin aliquot, polymerized for 1 hour at 37℃
for 2nd polymerization, added half of remaining tubulin to polymerized tubulin (final concentration free tubulin ~ 17 µM) took out 0.5 µL for squash.
took out 1 µL for squash, added 0.875 µL (remaining tubulin) as well as 0.625 µL modified growth buffer contained 0.4 µL Mg and 0.16 µL GTP, 0.065 µL BRB80
warmed all components for 1
2 min in water bath before adding final free tubulin 12.5 µM in 4 µL
incubated at 37℃ for 1 hour
Gave up on continuous polymerization because the Microtubules were still short after 3rd polymerization step.
diluted seeds from 3rd step ~1:100 in new tubulin solution
1 aliquot tubulin, 13.1 µL growth buffer(no dmso or glycerol) 0.2 µL seeds (15 µM tubulin) put in 37℃ for 1 hour, also put seeds back at 37℃.
Made MT20, MT100 with 100x diluted seeds and left overnight, also MT50,MT100 with non
diluted seeds left overnight

8-8
Imaged Microtubules made 8-5

8-15
Polymerized Hylite Tubulin, incubated at 37℃ for 30 min
Polymerized rhodamine tubulin 20 mg/ml with GMPCPP at 1mM (2.5 µL GMPCPP (10mM stock) in 25 µL GB), 37℃ for 1 hr
Diluted rhodamine GMPCPP seeds into new tubulin at 20 µM (10x dilution)
1 µL seeds and 9 µL growth buffer, growth buffer had just BRB80, GTP, Mg at standard concentrations.
Incubated at 37℃ for 2 hours
Diluted seeds did not continue to grow

8-16
Imaged seeds from 8-15 at 10:30 AM after incubating at 37℃ overnight. Seeds have not grown more and there were tubulin clumps
Polymerized Hylite tubulin in standard growth buffer at 37℃ for 30 min, made MT50 and imaged 4x dilution (MT200)
Repeated seeding protocol from 8-1
1 aliquot tubulin and 6.25 µL glycerol growth buffer, polymerized @ 37℃ for ~90 min (10:10
11:40)
New tubulin aliquot, added 6 µL standard growth buffer with no DMSO (23uL BRB80 + 1uL GTP + 1uL Mg). 3 µL new tubulin mixed into 5 µL old tubulin. Final concentration 15µM. Iced and vortexed new tubuli, separated 3 µL and placed that in incubator for ~4 min before adding
Incubated at 37℃ for ~1 hour 40 min (11:50
1:30)
Added 3 µL standard Growth Buffer to remaining 2 µL tubulin, iced and vortexed and pre-warmed for 2 min then added 0.5 µL of seeds. Incubated at 37℃ for 2 hrs(2:00
4:00). Left seeds and diluted seeds in incubator overnight

8-17
Followed the same procedure as 8-16 but used rhodamine tubulin for the first step and hylite tubulin for the second and third steps
hylite tubulin polymerized off of seeds and produced longest microtubules observed thus far

8-18
Reused seeds(rhodamine) from 8-17
First polymerized in 15 µM Hylite tubulin:
added 5 µL growth buffer(no DMSO) to Hylite tubulin aliquot
removed 3 µL more growth buffer to the remaining 2 µL hylite tubulin to make 5uL of 15 µM
added 0.5 µL seeds from yesterday
incubated at 37℃ for ~ 4 hours(starting ~ 9:30 AM)
Next polymerized with Hylite and unlabeled tubulin:
Combined remaining 3 µL hylite tubulin with 5 µL of unlabeled(50 µM) to create final solution of 46.25 µM
added 0.2 µL Mg to the new tubulin solution(unlabeled already has 1mM GTP)
added 3.5 µL new tubulin to remaining 4 µL of polymerized seed solution resulting in 7.5 µL of 21.58 µM tubulin. (note that we intended to have 15 µM but I made a calculation error. 15 µM would be ~2µL of new tubulin solution added to the diluted seed solution)
Polymerized again with hylite and unlabeled tubulin:
added 3µL of remaining hylite and unlabeled tubulin to polymerized tubulin (~5.5µL polymerized remaining). Final concentration of tubulin = 16 µM

8-19
Imaged Hylite and unlabeled tubulin Microtubules from 8-18.
Both MT20 and MT100 showed no visible microtubules, just large clumps of fluorescence (probably hylite tubulin)


Summary for September

In September we summarized and formalized our summer results. The abstract was prepared. We started working on the wiki website and wrote the script for the video. Lab Notebook entries were compiled and added to the wiki website.

Summary for October

In October we finished filming the video and created a PowerPoint presentation to present our findings. This wiki website was completed 10-20-2016.