oysters. Oh, we've got some of them in the cold water but they've been in the cold.
Where's the colon? So
you know what the minus 80 was always at the end of that hole. Oh, okay. All right couple of water tanks but they're just sitting in a bucket and water. Okay,
I just need some sea water.
Yeah How much do you need? 100 mils
we'll see Ya.
We're gonna have a Reese's Peanut Butter difficult choices
the rest of SEEBURGER stuff
Yeah. Yeah, I mean that was pretty much it after you're done.
Nice to have that all finished up today
alright,
let's zip anything they want to talk about up on top
with any major topic, we want to go over to the diet. Anybody got anything they're dying to talk about? Nope. Now we're gonna go straight to the NOFO competitions.
Last week, every week, every
week, come up with anything so what we're doing
right there, all of us want to talk about what you're up to now. Do you got going on
all the things? Oh, no, thanks. Yeah, um, so we've been at a point Whitney every week. We've started kind of a new phase. So last week, we went out and sampled the individuals that we had from our what we're calling the life stage carryover project that Eric was doing. And so we collected a bunch of samples there and we brought back some other animals that we're going to use as tests for a potentially a liquid respirometry assay, which is basically where we're gonna try today you put an animal in this liquid then in a solution that has a special reactive salt, the pH and you should theoretically be able to use a plate reader to color metrically estimate respiration rates. So that's kind of one cool thing we're going to try out. While we started this week, was we're going to start moving in both seed and adults to do some conditioning. The adults are going to condition just for one week next week hopefully we start that and start out planting those in parallel with point Whitney out planting other adults that have just been an ambient and then we're going to start conditioning seed as well for a month, two months, maybe depending on when we out plant those where we're going to bring them in the lab and do an acute, say two to five hour high stress once a week. So we're just gonna start those conditioning steps to see if we can elicit any stress hardening effects in an alginate hatchery environment. So we're gonna be starting to do that. And then we're writing a sea grant proposal to develop easy to use high throughput, low cost biomarkers that growers can use to estimate their stocks tolerance. So those are kind of the oyster things that we've started here in the last couple of weeks. Me and then all the coral things happening all the
time. Your question?
Oh, yeah, I was gonna ask more about the liquid respirometry assay. So that in that's for the seed, it's not for the adult.
We couldn't do it with any size of thing if you just put it in a larger volume of liquid, but we're going to start trying it today with like a range of sizes and see I mean, ideally in our context, we'd be using it where a grower could test their seed before outplanting to see their stress response and then use that to predict how they will survive in the summer grow up. So to do it with small things up to that like pre out plant seed size. Yeah. But if you put it in an adult in a big container of fluid, you theoretically could do that too. Yeah.
Is it kind of like the assay is it kind of like, like you have a color, like scale that you just just like visually compare. So
we're going to do it on plate reader, which is how the protocol currently works like a just a color metric plate reader, but you we could like depending on how well this works and how much of a color difference you see, we could develop like a color bar that a grower could just hold up to the liquid and match the color and that will kind of show you the rate. That could be an application but yeah, right now it's a plate reader method. Okay,
I haven't used the plate reader before so I don't know how that one works is
just like measuring fluorescence out of that fluid on an analytical instrument that gives you a precise metric of color as reflectance essentially.
You don't get a slope. Do you get like a rate is it like the number that fits out? Yeah,
yeah. To like, you know, take a measurement at the start. Right and then after 30 minutes, measure it that same plate again, and we could get rates we haven't really seen we just need to see if like we see a change in color. First. Do great then we could figure out well, how we could extrapolate that to a rate and then we need to ground truth that in some way with like actual oxygen consumption or something to Yeah, relate that back.
Well, really, no, it needs the color. So we need the color to be informative about the field. cares about actual lock? Yeah, I
guess it just depends on what it's actually what is measuring. Yeah. If you want to know if it's, it's pH reactive. So is it reacting to other things, or is it reacting? Yeah, so that part's a little tricky. But yeah, we'll try to see if at least we can get get it to respond to something.
Yeah, I guess that if it's pH dependent, it'll be like you wouldn't, unless you have like the same water, like pH and like other characteristics. You wouldn't be able to compare across only within, like, a group Yeah,
the right controls. Yeah. If you have a control, so if say, I mean, you'd like any other asset, you'd have blanks, right? So you'd have the pH of just water without an organism in it. And then that's your blank. You the difference and then we would normalize that to say, volume of the animal or side, like you know, you'd have to have a normalizer
you're gonna have to like take the like a dry or wet weight of your, of your, like, your I'm guessing there's gonna be like a couple of different little barbers floating around and each one does
have like one animal and each one wants each one. And like quick weight well, so basically we'll have a 12 well plate and then each of those wells is a mil of fluid. We put it, let it just hang out in there, till we see it. Basically. And so then you would just want to have the same volume of water in each sample and then do a what wait is a really like quick and easy way to normalize that right now. Yeah. So we'll hopefully have like more details depending on if that works. We should have had some blanks in our trays today.
Because, again, the
name is reservation reserve in we're not sure it was a reservation.
We made like commercial stuff that you can buy called LMR. Blue. Okay, which is basically the residents are in salt already pre dissolved and set kind of ready to go.
Oh, nice. So then from that you can make the solution is that the solution itself or even like diluted and actually,
yeah, you would just use that and add it to the media assets. Oh, so. Okay. It's actually mentioned in the in the in the repo. Oh, that's great.
Okay, that was I talked to Jose last week about the lab for the classroom team exporter. And kind of similar to like, Stephen, when you had us in that grad lab for environmental physiology. So like, we basically talked about a list of things that like the students could measure, and one of them was respirometry. And there's like this whole debate over like whether or not to get like a new fancy restaurant butter for like a whole department or something worth it. And I mentioned like this liquid rests was reservoir, reservoir, and thank you. And he seemed interested in that and then that would also be like a test of how percent crabs so I don't know if that would also Ross.
I mean, I don't think it necessarily matters to the organism it is, but if we just know that a change of color relates to this amount of respiration or something, you know, it just needs a little bit of I think there's been some ground truthing before but just so we can prove to ourselves that it doesn't we think it does.
Yeah, that'd be cool. Yeah, and it doesn't have to be airtight. It's because it's not an oxygen measurement. It's a pH. No.
The salt is taken up by cells and converted to a fluorescent. It's reduced to a different fluorescent compound that is a reddish color. Yeah, so it depends on the uptake by cells.
And again, to if you have a blank if there's any like, exchange or changes by air being open to the air, it will show up in your blanks. So that can be subtracted out. Yeah.
Kind of useful. Um, right now I'm trying to like remake all of the respirology chambers in the padega Mineola because they're like, not airtight and they're, yeah, it's kind of a pain in there. So this is you said there was a repo or your look like sort of like putting the other protocols for exploring using this thing. What's it called if I were to search it
ended up in our lab repo somewhere. Oh, is probably Yeah, I'll post a link for it. Yeah. So the airtight, airtight part for respirometry matters because you're actually measuring oxygen. directly measuring oxygen, so it doesn't matter in the same way. Oh yeah. And I've been I've got a bunch of quotes too, on respirometry equipment and stuff. So that's something we could explore to the the reason that we want to go with something like this that a grower could realistically use something like this cheap, high throughput, so that would be the goal.
To buy the respirometry for class and then we'll use that we don't have we can say for planning,
I have a $20,000 system I really want to
talk to John is something that's like actually useful to to like everyone else, too. So it sounds like there'll be like at least three classes per year that will use it and I think because I'm not for him, but if he hears that, like we're interested in using my class
to my class. Yeah.
Sounds good. Round,
either in different places, and
it'd be awesome. That's not much money for the department. To me, that seems like a lot.
In terms of like the teaching budget,
yeah. The two systems that we could look into are an str, which is essentially like a 24. Well, oxygen sensor that you put either two or four mil vials on top of and then you can measure oxygen for as long as you want. That's good for small things. And then you can also just get spots that you install inside beakers, jars, whatever you want that you read then with a probe from the outside of the jar onto that spot, and you could have up to 1420 probes running at a time realistically. So both of those systems would be about 12,000 each. But it's fun to have really good to have Yeah.
Then we should probably do a report of ours, like decide on the title and then I can start to email people for letters
for the grant. Yeah, I have a pretty important title. Yeah, we'd like to do we like that title that.
Wrap it around and say I say here and see what I
am. This is from chat. I put in an abstract and pet chat. Ducati give me a title, early life stage biomarker profiling for precision aquaculture, optimizing tools to predict growth and survival and hatchery stocks. That's the title of it again. I mean, it has some buzzwords in there. It's got
Yeah, I'll look at gonna see like, maybe drop a word or two at the end say Well,
early life safe biomarker profiling for precision aquaculture, optimizing tools to predict growth and survival in oyster hatchery stocks. We can probably say in oysters,
yeah. Or yeah, that sort of thing that you can either say, you know, what's yours or in growth and survival or just to
predict oyster growth and survival. So yes, that is ready to send out whenever you want shellfish.
Yeah, got it. I would
change it to shellfish resilience, how about that or something? Because that's what we're doing. We really don't grow. It's it's really not our concern, per se, what
we're really I think they care because they care on a production standpoint. Okay. But I mean, we obviously make the case that resilience equals production. Yeah. But we could have to predict shellfish production and resilience.
We can type some more athletes. Yeah, sir.
being finalized today, and then you could send out some meters.
Yeah, just like one word at the end there to kind of sum it up. But yeah, it kind of depends on the RFP tools of the whatever it makes.
Yeah, we can work on Wednesday.
Yeah, we'll keep you all updated.
On all the things and then yeah, I'll say here's a link in Slack to the protocols if you want to look at that more. So I guess they're dropped off
others All right. Thank you
What do you got going on, Sam?
Yeah, I guess over the past week. Our board stuff renew SSL certificates for the two servers that had expired, and that was an issue that was triggered by renewing those certificates and resolve that issue dealt with a bunch of annoying procard reconciliation stuff. I read ahead for the tenant sweet grass stuff for next week. So that run discussion stuff tomorrow, maybe or early next week. And then yesterday, spent a lot of time working on see bigger manuscripts going through cleaning things up. Helping highlight which sections were written and which code corresponds to which things have been written and results and vice versa and starting to organize the repo or more publication organization, meaning I putting script highlighting scripts that were actually used in the manuscript and sort of separating them from those that were not used in the manuscript and considered more exploratory. And then today working on this roseburn assay, and it's essentially going forwards and qPCR RNA solutions are more than residence Earth stuff.
But it has to do with all the bottles that Noah dumped. Those are the waste removed.
It's not rude. Yeah. Okay, well, what
about all the bottles? Can we throw those and what can we recycle them now? Yeah, I mean, we added I mean, we shouldn't throw
them in the breakable glass boxes. So just save some big boxes. Get some, you know, heavy duty garbage bags, which we have and tape them up and label broken glass and then they can go in the dumpster. Alright,
maybe we can assign that to know, maybe an issue or something. Yeah.
We're right on if you think we should be here as long as one was enough waste. Well, yeah. There's might be some still I haven't looked at all of them, some of them kind of pride in touch. Some of MAXDOP are excellent as
is there equity I go along? Well, I started like my time crafting drywood Washington seeker and so that was like a lot of orientation. A lot of like stuff on the Washington secret inside that I just assumed it would be exempt from but I now know everything about that dilapidated old building over there, and how to open doors and wherever it is found. I also want to like a quick little inventory of their freezer room in portage bay, which is under it's in the basement. It's underneath a gigantic event. You have to duck down half the doors covered by the van. And like it seems very It seems illegal somehow. But it's definitely secret room that I now know about. So that's fun. I've been like helping them put together some of their like kits for teaching volunteers about like identifying crafts, remotes, lots of planning stuff. That's been like a little more time consuming than I know that it will be in the future just because they're trying to get me on board with some stuff and then also to go to some of these in person trainings that are coming up with them. So next week. I'm gonna go out into the field and we're going to be collecting some global logarithms putting some new ones out. And the day after that I'm going to Port Townsend for their in person Monitor training, which will be really fun. So I'm excited about that. Those are really the only two big things like have that next week, which is two whole days but they only do like five of these trainings or so like a year in person. So that'll be like the biggest time investment of like, this quarter slash next quarter, to just go to a couple of those. But it's very tempting to very cool. It's cool system science program. And it just seems like they have tons of data that no one's really using, or training. So maybe that's something to
the effect that they wouldn't let you use it or some kind of. Yeah,
I think so. Here's, here's what I think the line is drawn is that if I'm doing something that I'm getting paid for, it can't be like a research. It can be research.
So I think once you're done work, you could be research
once you got exactly so it's like if I'm not logging those hours, I think it's gonna be like a discussion we had because they do have they have so much stuff. It's really interesting to see like the they just have like tons of like frozen crabs. So there's got to be something just kind of
literature on on just invasive species and epigenetic striving that,
you know. Yeah, that's really cool. And I know that's like that's like on the backburner discussion for them as just talking about genetics in general. Yeah. I don't know if they've really gotten to that. Yeah.
Now just identifying, you know, killing them. Yeah,
basically, well, essentially, like the bigger trapping efforts are done by like more local national agencies. So we just like, try to find them and record them and let those agencies that are affiliated with those locations now and then they do bigger trapping efforts to to get rid of them. But that's going well. It's been pretty interesting. I've also been working on my application for the application Science Center fellowship, all my ducks in a row with like the the partner piece of that with Mark. I've talked to Eric who's helped me with the proposal itself with content and then Steven, I'm gonna send either tomorrow or more likely, like Monday morning, just the letter of support for that. So I'll be in touch about that. The whole application is due Monday, but it's like the easiest fellowship application or it seems just great. So that's going on. And then I've been actually working on my SEEBURGER related manuscript stuff. So that's definitely fallen off a little bit over this last week. But today and tomorrow, I'm not doing anything related. And I kind of protected these two days like no matter what, to just
grab, do you think it's worth it? I mean, is there ever been a case where an invasive species that you've tried there, somebody's proven the eradication or identification process has kept us busy though, I'm assuming they must have some data on that but
I think like there have been some successful, like, population diminishing eradication efforts, but I'm not sure that there's like a true case of eradication, working well, with role
and all that but yeah, I just can't imagine that just do anything with a marine larval stage. Just like to get slowed down, but
it's interesting. Yeah. I think like, the way that they phrase it and like our webinars to the public is like they're just trying to slow it down so that they can work on protecting. Okay, that makes Yeah, which I think is nice. Yeah, because all of the like eradication efforts I can think that's that like we're successful or like, plants are like, Yes, I still think it's a little more control over.
But yeah,
cool. super interesting.
Yeah, that's really a loser Are we just we're not we don't know what you hear because they've never tried crispy yak and Chris, what do you got going on? Anything fun?
Depends on how you define fun. I got the nature opinion draft out yesterday. I needed to get it out yesterday. So that is cool. And I'm terrified. Because the first draft can be up to 1500 words. Mine was 1300 and they're going to cut it down to 850. So I don't know how that's gonna go, but it'll be fun. I'm working on the biomarker data. That is kicking my butt right now. It's yeah, not this week. But next week, I will be in science hour and then I'm sure I will open issues because in order for me to get this draft out by the end of the month, I'm going to need a lot of help. I just have to get to a point where I know what help I'm specifically asking for. And I'm hoping to have that answer by Monday. Working on the agenda for Innovation Grant, because I'm an idiot and schedule that meeting today, because why not? I get on a plane tomorrow night to Pakistan. So I need to get some things knocked out. Finished in a quarter of course with the TA ship. next quarter. I opted for one that's less student intensive. So that gives me a significantly higher amount of time that I can do the things I need to do. I need to follow up with you. And Kathleen, I think about what we want to do with this shellfish experiment, or if it's an option. And also, I believe, I have an idea of what my site choice needs to be for the epigenetic work, because I do have enough visualization in the biomarker results. That I can see what I want to do. And for that, what I want to do is look at the differences across sites with high p 450. But not high so d a site with highest so D but not high p 450. And I control site and potentially one that has high both just to see if there's some sort of response individually across those sites. That makes sense. And then from those four sites, maybe, I don't know, one to two muscles for each site to muscles for each site. I don't know what number would be relevant based on
what we're gonna shoot you need to shoot for we're gonna do it. We need to go higher on replication as opposed to die like suicides Max,
and then two sites Max.
Probably, unless where the money has been to where the money's coming from, but yeah, I mean, you're gonna see I think you're gonna see too much variation in individuals to get a statistical difference unless you at least how I mean I could maybe can be convinced five or six muscles, so that would make that would be like twel. Right if you had control and treatment,
okay, so if that's the case, then I would like to use the PIN code site as the home site, where we have the reference muscles, everything comes from there as the clean site quote, unquote, and then I will pick a high p 450. site and a high sed site. I want to see if there's any difference in relation to biomarker expressions. Because there two different biomarkers that are testing two different things. So I think that would be more relevant. So three sites, two per site, three per site. This year, I'm sorry, say again.
That is what that involves this year.
So what I want to know is if there is comparable or different gene expression or different epigenetic response to the contamination load or the that I'm sorry, let me be more specific. I want to see if there's specific changes in gene expression, through the mechanism of epigenetics, to see what if these biomarkers are triggering the same stuff or not? That's really what I want to know. Are they are these muscles, persisting? Are all the mechanisms same doesn't matter? Does it matter what kind of stress we're stressing them out to they're gonna respond similarly, or are they not? And then if it's can be linked biomarker to gene expression can be linked, then that's a different conversation later. I don't know what that means.
Very simple of the audits is is that contaminant exposure will alter the DNA methylation pattern in muscles muscles, right? Yes. Yeah, so maybe we do a piece hair piece wise pairwise, step by step. When we do just test that control and highlight expose and then we want to get in if there's no effect, then there's no reason to do too, but there is an effect then second stage is look at the look at another contaminant. And see,
when we're saying highly exposed, does that mean my site choice should then have one that's just highly exposed? The highest one that the one that shows the highest expression for p 450 and sad then
whatever others this is, I mean, unless you have other data from my chemistry to know what the contaminant load is.
We have Yeah, but we have some whole muscle tissue data. And then we have the water data is from 2019. But I mean, the muscles were out planted at 2021. It's not that great of a difference. These are, you know, persistent legacy chemicals. They're in there for a reason. So if I'm just then looking at contaminants I'm sorry, DNA methylation, in response to contamination that I need to pick Penn Cove and my spot that has my highest expressions, generally speaking for both because if it doesn't make a difference, like I just need to see if something is is triggering a response. Then I can go back and dig and say what was it?
Yeah. That makes sense. Yes, that makes sense.
Okay, so then I will put that in that issue so that we can close it and move forward with that because five granted that is staffs research money that has to be spent sometime before next month, the end of next month. And then use it you would pay for whatever the difference was, when that was done is it's five grand so I don't know how much that'll go for but yeah. Okay, I will close that issue. And then we still have to sort the other selfish issue. Oh, speaking of I attempted to use my API for the GPT in line in source, whatever, and it is either expired or not usable anymore. So is there Do you have a link somewhere where we can go back and get an API key? Because I can't find it. I don't know what I did with it.
But it is true.
When I submit things that certain hours a day you don't see them so no, I didn't Yes,
mash is always gonna get done. Okay. All right.
I will do that. But that's what I've been working on. I'm going to end up having a five male one female cohort for Doris Duke. So I'm just trying to figure out what the hell to do with that many guys in one space. I live in a woman centered world I don't even know are not just doors two doors do again the RU I've got two R Us and four DDC SPS. So I don't know. Going to hang out with boys on an island. Matt will be excited for once he won't be relegated to like the sad cabin. He'll get to sleep with the boys and I'll have to go to the sad cabin now. So it'll be good. It is excited. Well, it'll be a guy dominated year but yeah, that's what's happening
all right. Sarah, what do you got? You got you kind of give us a little sneak peek about what you're up to repairing spirometers anything else? Monitor or less? Yeah.
Yeah, it was actually really helpful to listen to Chris to talk about because I'm also like working on figuring out sampling strategy for experimental design. So I've kind of been out scouting late at night, low tide for aggregating anemones. And I found a bunch of different sites that have them. And now it's kind of up to you know, for my experiment, I'm trying to do a simple like two by three factorial design with two different temperatures and a control and a low dose of microplastic lead shape, and a high dose of microplastic lychees and looking at, like multiple responses to multiple stressors to trying to see like, how does that like, does it affect their respiration? Their microbial communities, their gene expression. So those are the three responses that I'm trying to measure and photosynthesis of their symbiotic. So four main responses. And my hypothesis is that it's actually kind of shifting because So Jackie teaches a class with like four pins. I don't know if I'm bad with remembering the numbers of the classes, but it's tropical marine ecology. And her lab, her whole undergrad lab, basically used the app Teva anemones, which are these tropical anatomies that are used as model organisms for coral research. Because they're tests for like aquaria they're like, super easy to take care of and really resilient. And they can have some bonds or they could not have to buy off the kind of similar to the aggregating in them many that I'm going to be working with that is like local to Puget Sound. And they all had like, increased in density under heat stress, and microplastic lead shade stress. So she kind of like did like a pilot in her lab. And they had like increased respiration, like increase in density, like increased photosynthetic efficiency under the multiple stressor, like positive positive movement group, which was like pretty interesting and like the opposite of what I was going to hypothesize. So I'm kind of like, thinking about that and digging into the literature again, trying to kind of like understand why and I think a lot of it has to do with like, when you leach something out of like, like plastics have, like carbon backbone, you know, like, and then there's like all these other like trace, metal, those things that are kind of also in the cocktail mix. And so when you give synbiotic increased heat and increased trace nutrients they can take off. So the symbiotic density and like increased photosynthetic efficiency and increased respiration could all be related to that kind of like combination of it being more beneficial for the symbiotic but maybe not for the host. So there's kind of like host environment dynamics there that like, are kind of interesting. Long story short, I'm trying to figure out like, do I select from multiple sites and add site as a factor? Or do I keep it simple and like collect from all one site? If I did that, would a reviewer be like Oh, this isn't representative of like, a, like greater population could have different genotypes.
You could argue that even if you had if you had two sides or three sides, you could argue it's not representative. So if you were to add what biological hypothesis, would you be testing by site that would justify adding another site?
Yeah, my question isn't really about site. It's more about like the organismal forsan. But different sites have different like characteristics, like Chris Chris's project, some sites might have like, more influence of like past history of like pollution, stress, or others are more pristine. And so like, if I was to just pick one site to do it, I'd probably pick a pristine site to see like, because I don't want them to be primed for the stress before seeing like, well, what does it do to them for the first time and then the aggregates are, you know, they kind of like it's there. They're clones. But they cover they're like maps of anatomies that cover like an area or rock or boulder. And so if I were to take all the anatomies from one mat, I could assume that those are clones of each other. So do I use all the same genotype for the whole experiment? Or do I take maybe like, seven or 10 different maps and have different genotypes and then have one in each treatment or like multiples in each treatment? So a lot of I guess if anyone has resources for, like, experimental design that they have found useful for making those sorts of decisions. That's kind of where I'm at right now.
Yeah, a good place to start to for that is if there's any existing literature on just variation of any response between genotypes of the species that you're working with, because if growth, survival, any physiological response varies quite a bit between individuals, then that's a pretty strong argument that you'd need to include multiple distinct individuals to be able to characterize and be representative of a population level response and being able to follow you then go into the question of, okay, do you have, you know, one of each genotype in each treatment, right, which there's a good argument for doing that, to capture that individual variation and account for it statistically. So that would be a good place to start is like, what's the expected variation between genotypes? Okay, yeah, so more information.
When I guess when we talk about like, it's like variation between genotype like, I feel like when I think of like, GE, like, like gene expression, like it's kind of, I can visualize the heat map, but like between one individual and another How do you like quantify the variation? As he like, yeah, like, I think it would be like per gene, right. It would be like, how much expression is is detected but across the board that's kind of like, I feel like harder to quantify. Maybe there's like some math wizardry.
Correct me, but I think generally, you're, what she's saying is that, looking for studies where they look for two different populations are good, and you might not have this luxury and all species are from two different locations that are presumed to be different genetics or background to see if you can if their response their responses in general, no matter what, whatever parameter you are, it's affected by that like it not everybody does that. So you might not be able to do it your species. But like if we were to take and we've done this, Olympia oysters from three different populations and measure their growth rate under different temperature and berries, so that gives you the warning that like anything you want to extrapolate about Austria learning in general, you have to be just aware or you have to give that caveat that it's probably it could be driven by genetics. Is that what you're saying? Yeah, and
like in any like in all of your coral studies, you would have done the same thing right. You would have said we know that each colony is going to be different because genetically, they're different. They're going to respond different to things. So you take a fragment of each colony and have it in each of your treatments. So you can go back and calculate how does that colony respond and use that individual variation of say a random effect or something to say we know that individuals are different, we're going to account for that noise given that we're seeing this root level difference. So maybe that you need to do the same. I would expect you need to do the same thing here. Yeah,
I was like kind of thinking I would take that same approach, but I wanted to take a step back and make sure I was Yeah, because because at the same time, like they're clones, but they're not a colony. So there could be even within an aggregate mat of clone. There could be variation. So then I was like, do I take like, a couple from each aggregate and have a couple in each tree like three in each treatment from each aggregate? And that's just it gets hard because then as soon as you start adding in more like your sample size blows up pretty fast. Yeah, but anyway, well
to get a biologically functional, same unit and everything right, even within a coral colony that we're more familiar with working a piece of a branch is going to respond differently than the piece of another branch on the same colony, right? So it's just you want to be able to statistically control for as much of it as you can. And one way to do that is it whatever your lowest operational unit is that you can work from a math a colony, whatever it is, you have that represented in each of your treatments, so that if one colony or one mat always grows more than the others, you can statistically remove that variation. So kind of have to Yeah, at some point, there's like you have to make your best guess.
Yeah, so I'm guessing,
thinking about so yeah, it is. It's a balance between adding more things and sample size as we were just talking about Christmas stuff.
Yep, so that's what I'm working on. I'm going out Friday night. There's a really big low tide on that really big. There's a low tide Friday night at 10pm. So, yeah, that's my plans for the weekend. Exciting. Does it make fun? Yeah, they're kind of hard to get off the rocks, because they're such down pretty hard with their fetal disk. And like I've been trying to, I was trying at first to like, chisel them off, but that kind of like cut their tissue. And this is terrible. But like, I found that the best way to do it is just to like, press them with like, the fleshy bit of your finger for like five minutes and then they pop off. But it like takes a really long time. But their fetal disk is like intact and not like scarred. up or anything and then they re adhere to whatever other surface you put them on after that. So I am soaking in enemies all day. Which, you know,
exactly what people think science is. So that's great.
I think it'd be really cool in your gene expression data. Because I I'm just really interested by the response that you they saw in the preliminary tests, like cool to look at all of your central metabolism markers and stuff like that because you'd expect that as a symbiotic grows, and is more selfish. They give less photos and fates to the host. So it'd be really cool if you see something like that.
Oh, and that would be visible in like, total RNA medical, or that's
you'd see signature signatures of the in tab along with gene expression, like whatever your indicator is, you would expect to see an increase in genes related to catabolism of lipid reserves or gluconeogenesis versus genes that are responsible for metabolizing glucose, for example. It comes straight from the Senbei insert glycolysis. So just kind of fun things to like, think about as you do this, like what you do, but that's what I would expect based on the results Jackie was getting.
Okay, cool. Yeah. Thank you. I'm sure we will talk a lot more exciting.
Less What are you been up to?
I've been up to having Sam helped me through this GitHub issue to decipher all that I've kind of done with this qPCR work and how to move forward as well as with your input, Stephen. Thank you. Yeah. So the responses have been really helpful and it's like nice to get everything kind of organized. So it's like what I've done because I've been teaching so much this quarter. I haven't had that much time to focus on like work like just like working through this, especially because I was kind of at a loss of like, how I should move forward without wasting any more resources. But I think the suggestions that Sam made recently, good work in terms of like, just trying it again, but not with like, all of my concentrations of cDNA I think that sounds like a good idea. I think that yeah, so yeah, that's the only question. And maybe I'm a little bit overconfident, but I think that like if the technical replicates for the housekeeping genes are like, perfect and for like everything else they're not. I don't feel like it's like a user thing, but rather like something else. going on in the reaction. Can that be a possibility? That's something I like looked up on like ResearchGate and some people said that that could be a thing where like the technical replicates for the use whatever for standards don't look the same because of something else going on and they were action.
Can you Can anybody here is I can hear you know? We try to talk while you're talking? I guess it blocks us. Yeah. Let's stop you like a long time ago. Oh,
well, for one quick one question is why are you not doing RNA seek? Why are you doing qPCR? That's the first question.
Ah, money, mostly. But if you're gonna, you're, what's it?
Like? Do you think it's gonna cost you a degree? qPCR?
I would assume so. I mean, there's just like, there's just like a certain budget allocated specifically for like, cell like using it for cell culture or RNA seek or using the RNA seek budget for the cell culturing aspect that like, this is not that so that's why I move forward with qPCR. Yeah, that's where I'm at right now. And then, yeah, go ahead. Curious,
out of my own curiosity. Do you know how much qPCR is costing you?
At the moment? I feel like not entirely, but each cyber green bottle is like 500 bucks. So that's not using more resources.
How many samples that that allow you to process?
Um, quite a few honestly, I think I calculated and I only needed like maybe two to three bottles if everything went perfect. But things aren't so perfect. Right now. So I've been trying to like repeat like the standard curve situations because this is I think, I've like ran a total of like 696 well plates, or five. So
okay, and it curves there's no reason you should ever run a standard curve.
I think that there is just one more because you because you need to like for Okay, well off the top of my head. I've always ran a standard for before qPCR because I think you need to like show that your primers like work well across the board for like all your samples. I think like you need to get that efficiency value before moving forward with the rest of it. Like a delta delta, whatever. qPCR
No need a standard curve to get efficiency values. You don't so, so yeah. So there's a lot of things. I mean, I did you look at the handbook. I mean, you don't, I would just I would I don't I'd hate to see you waste a lot of money. And that's because there's a lot of stuff going on and a lot of stuff being run and I would go about it a different way, like first every I mean and you're welcome to come up here and do it and but I mean, every time like you write you get a positive and you're no template control, which I think you're getting then it's like you just store everything out and you can't really go anywhere from there. Right. And then I guess what I'm saying is I think you're making it more complicated than it needs to be which is going to be which is going to run through your reagents at a pretty high rate. And that question is like it a certain point you're going to you're going to hit the sweet spot of RNA seek but I mean, that's a separate issue if you wanted to keep it at that time, but I think I think I think I would go I would take a more simpler model for troubleshooting. I don't know I mean, Sam, who's had his head in this more more than that. I mean, what do you what is your take Sam?
Yeah, I mean, they posted this morning. Just, I mean, just test out your primers, not with standard curves. Just test them out and see if you can get clean MTCs and see if you see single peaks in the milkers Yeah, and then if you do then those primers are good. If you don't want the others then either redesign new primers or just forget about those genes.
Yeah, I think that's I would agree with that. 100% that's that that's the most effective way to troubleshoot and not and not go through, not shoot through the reagents. Because you might better if you're really dead set on one gene, it's much easier to just design a new primer. Then make it force it to work. Because it's okay. It's quite possible that it might not ever work. If that makes sense. Yeah.
Okay, well, yeah, I guess I was at the point already that I wanted to order new primers anyways, but was concerned that even if I ordered new primers if it really like an issue with the sample itself, like if I could just resolve it now, before moving forward with that,
like my firt my first take is it's a it's not a sample issue. I don't think I've ever had a sample issue at this point. I mean, I can't imagine it ever being a sample issue of a one off. Can you matter? Um, can you what are the chances of being a sample as you Sam, I
mean, I feel the same way. We've never counter sample issue. Yeah. Okay, problem qPCR, especially since we use the kit for isolating RNA and whatnot. So things should be pretty clean. Yeah,
and you clearly there's amplification plots, even if it's contamination is you've got nice amplification plots. And you've got at least some nice Mel Mel curves.
So I think your bio, so I wouldn't be concerned about your samples. I would just do like what's at play, you know, I guess whatever is less and less comment since you'd like that would be exactly how to approach it.
Okay. Men, have this just sort of a initial test before you go into the standard curve stuff is in case you have genes that are going to be lowly expressed. That will also guide you on how much cDNA you're going to need the input in order to be able to potentially detect expression at all. So I know there was at least one gene that you noted came up super late. So things like that. You can evaluate now and say, Okay, well, you know, using one microliter of cDNA I'm probably not going to detect expression for most of my samples for this particular gene. I might need to bump it up to two or three my religious CDA in order to actually get usable values for that particular thing. So just doing some initial testing informs you with that those decisions as well
Well, that's pretty much all that
I have going for me right now.
Are you done like playing with playing with animals organism?
Not quite know. I think I'm pretty soon I wanted to just like run another exposure and save the samples and said we're like ICPMS to measure bio uptake of the nickel chloride relative to like, what's in the water, but I've been waiting until they're back in season so that's sooner when it starts warming up.
Another another element of this qPCR RNA seek is the qPCR is going to take a lot of time. I mean, you already have RNA isolated. You could take a half an hour to package them up and dry ice and send them off and that data back in a month. And you can do stuff while that's all happening, you know,
what's it What's your experimental design like? Do you have something simple like nickel and iron nickel or something
or it's mostly simple, I have two concentrations of nickel and then a control. But I think similar to like what we're discussing general right now about like them being really different like these animals. I also have to control or like bin them by blast genic stage because a lot of the stuff that I'm looking at are a lot of the genes I'm looking at. were previously using the trellis to like look at each of those developmental stages because they like undergo apoptosis every week. And like nickel chloride will also induce apoptosis. I have to identify that. So there's like 12 missions I think, technically if include stage. So
same review, kind of matter we do anymore. We're never going to do an amount of methylation job on the con. I don't think we have anything in the cube to do it because they we can even throw some on there. Sneaking around but I don't think not at the moment we don't.
Right anything else?
going on this week? What's what's later today? Thank you
so yeah, so next week I'll be I'm gonna be at this salmon. Meeting in Seattle and then the next week I'll be in Charlotte, but then my sabbaticals?
Yeah, tell me about
you're gonna give us like a presentation but
what I did on my slideshow I just put Chris in Slack, how to get your API log in. Is that right Sam? You can log into open API.
Yeah. Good. That's the part I didn't know either. Yeah, like,
I didn't remember either. I can because I can log in and see that y'all were all in the organization, but I can't you have to get your own key. And if you're not part of the organization you want to be let me know and then I'll go into my settings. And open API and put you in there. And then you grow. Then you go, you have to go into your own account and get your set an API key is that the name of an API key? And like I said, one of these lab meetings will, maybe two weeks from now it's on the calendar. I think it is the live calendar. to kind of demonstrate that so if we haven't done it before we last year, I think we played around with showing these different arm tools or plugins, where you feed it your your feed it you You give it your API. And there might be other use cases too. Chris, was there a different use? case that you were wasn't doing working in our studio?
Yeah, it was in our studio because the other API key issue I had I found a workaround, even though it's not working right now, which is why I need this API key to see if it can help me sort what my problem is before I start rebuilding from scratch.
Yeah, but I'm sure there's I mean, I it's unbelievable all the different things you could do with that I can log in and see all the stuff it's pretty amazing. But I do have I do I do have a limit on us and became your cable. Is there too much money?
Yeah, so we'll try it out. We'll try to show use cases for that. You know, probably two weeks from now. At the airline meeting, that I guess I Well, maybe not because I'll be flying. This is on a Thursday. Yeah. So. So we'll see.
Okay, based on your schedule, you said you're out next week. Well,
I'm in Seattle, but there's a salmon international salmon meeting in downtown and so I'll spend I'll be at that several days prior to the hatchery one day and then I fly out on Friday to Charlotte.
Okay, we have a meeting on Thursday. So I'm just going to cancel that. And I say this because I'm going to be in Pakistan, which is 12 hours ahead, which means I'll be like midnight when I am having that meeting. And I'd like to know in advance if that's real. So I'm gonna go ahead and cancel that.
No, all right. Well, thanks everybody for joining online. Have a good day and we'll see other night
