gene|fish

There isn’t much to report on the octopus mucus front. I’ve been out of town for a week in beautiful Bend, Oregon, and progress is still slow because of a particularly exclusive freeze drier. After my last 2D gel it was pretty clear that the new mucus samples need to be concentrated further, so we’d like to get them freeze dried before we attempt any more gels. In the meantime I have been attempting to read up on senescence in general, and senescence in cephalopods specifically. It’s tough trying to find helpful information on the topic. At first my project was going to be looking at protein markers for stress in the Giant Pacific Octopus, but after a miscommunication with the aquarium was resolved, it turns out they are really interested in senescence. If I remember correctly, the aquarium’s final goal is to be able to remove digested octopus from a six gill shark’s stomach and test it to see if the octopus was senescent when it was eaten. Pretty cool. That’s a long way down the road, but it will definitely be interesting. I was also thrilled to see that my mug shot from FISH 310 is now on the internet! Thanks Steven!

Rachel

V. tubiashii preys on the oystera and other shellfish larvae in the West coats. This bacteria undermined the Northwest prized oyster suppy, killing billions of young larvar. An explosion of the microbe laster summer shut down an Oregon shellfish hatchery that is one of the largest on the West Coast. So if you are one of those who goes to the restaurant to eat oystes, expect that the prices will go significantly up. Additionally, V. tubiashii is thought to be responsible for the disappearance of the generations of wild oysters from Willapa Bay on the southern Washington coast. Here is the information that I found online and think it is interesting: “Marine Vibrio species are known to produce extracellular products, some of which are known pathogenicity factors. These toxic proteins include cytolysins, proteases, lipases, siderophores, exopolysaccharides, and effectors delivered via type III secretion systems. In V. tubiashii, several secreted proteins, including a low-molecular-weight ciliostatic toxin, are suspected virulence factors in shellfish larval vibriosis. The east coast V. tubiashii strain ATCC 19105 has been described to produce a cytolysin/hemolysin (20) and an extracellular protease. ” At the same time I found out that V.T makes it home in the waters with low oxygen level. However, with the cllimate change, as the temperature of the water increased, they found a nicher feeding on oysters.

Today, I did PCR on V. parahemoliticus, V. tubiashii and V. vulnificus using differnt primers. This was done to check the spesificity of the primers.

At the same time, I did reverse transcription of the RNA isolated from the VT that was grown in the presence of clam foot and VT that was grown with foot.

Tatyana

OK everyone! The IEF is underway! I will provide live updates throughout the process (and maybe some pics, if possible). Check back in the post for the updates…

UPDATE 11:30AM

First stage of focusing almost complete. Here’s a quick pic I snapped of what things are looking like:

Looking good, I think. No sparks! Yet…

UPDATE 11:45AM

Second stage of IEF is almost finished. Things are looking real nice! Rachel’s samples are the two on the right and show no signs of scorching/burning/flames of any sort. The last run, however, I never even saw any banding of the bromophenol blue, so this is already a million times better. Here, look at the pic:

UPDATE 12:10PM

The previous stage ended successfully!

Now we’re to the final stage… 2000 VOLTS!!!

This thing is totally maxed out!! That big black dial in the middle sort of reminds me of HAL from 2001: A Space Odyssey. I can hear it. “HAL, open the pod doors…” “I can’t let you do that, Dave.” Creepy!

At least the dial on this power supply doesn’t pulse red, have an actual eye or speak to me. It’s bad enough just thinking “What if…”

UPDATE 12:40PM

Ta da! Success!!

Alright. Removing salts from octopus mucus samples is clearly the way to prevent fires from breaking out during IEF. And if you’re wondering about the differences in migration of the dyes, I have no explanation. I called Invitrogen and the dye is simply present to verify that current is flowing through your strips. Since the dye has been migrating since we started, we know that was happening. However, Invitrogen says that their IEF voltage “program” should result in good focusing, so no need to worry about dye migration. Sounds good to me!

Later in the day I’ll have some photos of the developed 2D gels…

UPDATE 2:20PM

OK, the troop has arrived! Here is a sweet action photo of Rachel returning the IEF cassette to the shaker with new buffer.

So, after she made that change and we sat through a short incubation period, we transferred the IEF strips to regular SDS/PAGE gels for running our samples out in the second dimension. Check the pic:

UPDATE 3:50PM

Gels are running and look beautiful!  ~45 minutes left for them to run and then it’s on to the staining, which is where it really gets interesting!

UPDATE 6:55PM

Well, things are wrapping up here.  We’ve done some staining:

And, we now have some developed 2D gels!  Unfortunately, Rachel’s gels proved to be virtually blank AND somehow they’re saved in some weird format that I can’t get to convert to JPEG.  So, you’ll have to see them at a later date (except for The Boss; he gets them via email immediately after this post is finished).

My two gels are below, first is the control and the second is the treated.  Very interesting!

So, there you have it!  All in a day’s work.  Easy, breezy.  Hope you’ve enjoyed following along!  Look for a “movie” later this week that flashes between the two 2D gels shown above.  That will allow for a bit easier identification of differences between the two samples.

Samb

Since the last time I posted (with my final protein gel photo from the first batch of octopus samples), not a whole lot has happened, with the exception of the IEF fireworks of course… My time has been spent prepping for 2D gels. Obviously, the first didn’t go as planned, but tomorrow (Tues) is my second attempt. We decided the problem with the last sample was salt. After going to the aquarium and watching how they took the mucus samples, this is not surprising at all. I’m sure the raw mucus samples we received were completely full of salt water. Prior to this 2D attempt the sample was filtered in a centricon tube, which will hopefully solve the problem. It will be interesting to see how the second batch of mucus compares to the first. I’m crossing my fingers for tomorrow, and will be selling tickets at the door before Sam cranks the power up to 2000 volts.

Rachel

After reading some of the posts, I realised that I can write about anything even if it is not related to science. But lets start with science. I have done many interesting  things since I started in this lab. I should say that I am not a PCR fan since you always have to worry about contamination. (and even if you do your best to avoid contamination, there is a chance that you will see unexpected bands in your negative control). Another thing that i have done is isolation of V. Tubiashi proteins from two differnt samples( one V.T. grew in the presence of the foot tissue and the second one just in the broth). Then I ran the isolated proteins on a gel. I also learned how to do Reverse Transcription.

Today, i isolaed RNA from V. Tubiashi that grew in the presence of hemocytes. Two controls were used: 1-hemocytes without V.T. ; 2- V.T. without hemocytes. Also, mRNA was isolated from the QPX tube, I should check with Sam what was in that tube. He just told me to do that tube as well and I did. I should say that all lab work is time consuming. You can spend up to 4 hours just to do one thing. I did not expect that it is like this. But I should say that it is much easier to learn in lab setting than in the classroom.

Thanks to Sam for all his help.

Tatyana

Hey everyone…..so i am being told to share the knowledge! My expierence here in the lab has been great. Recently I have been running gels with V. Tubiati cDNA and it shows differences in temps from 12C to 21C. (I hope this is making sense?) Today, I ran another gel with again the V. tubiati cDNA using primers from a different species and the results showed no bands (negative result).

Yesterday, I recieved 5’ and 3’ RACE sequences from Sam and ran them through BLAST to find the common vector sequences. Once this was done I crop the 5’, 3’ sequences of these vector sequences. Then I took these new 5’ and 3’ sequences and assembled them. Extracting most of this sequence we then assembled it with our known Prostaglandin sequences to create a final product.

So there……..Have fun with the new knowledge gained

:0) Christin

OK, first of all, two posts in a row by me referencing horrible ’80s songs (“Hot, Hot, Hot” by Buster Poindexter and “The Heat is On” by Glenn Fry, formerly of The Eagles). This has to come to an immediate end.

Anyway, the lab is significantly cooler today than it has been all week, but that doesn’t mean things in lab won’t still spontaneously combust:

What we have here is an isoelectric focusing (IEF) cassette with four IEF strips. Two of which arced and then ignited when the voltage was turned up to 2000V. Yes, there was fire. Yes, it was scary. No, no one was hurt. No, no equipment was ruined (other than this cassette, which is disposable anyway).

Important lesson learned today. Samples with possibly high salt content (e.g. octopus mucus) should NOT be run at high voltage.

Samb