It sure has been a crazy couple of months! I have been hard at work completing the data analysis on my research, making figures, and writing. I have also been trying to finish the molecular component of my research. Unfortunately, when I got my initial sequences back, there were nine of them that did not work. I tried troubleshooting a few different ways with no luck and spent last month trying to clone my PCR products to see if that would work. However, since my writing deadline is approaching, figuring out my molecular sequences will have to wait another month so stay tuned for info on that!
Check out what a good sequence result looks like versus a bad sequence!
Hey everyone! So you may recall that I went down to the University of South Florida before the holidays to look at the chemistry of my seaweeds, and I am happy to say that it was a great trip! With the help of the students in Dr. Baker's lab, I was able to run extracts of my seaweeds on an instrument called a Mass Spectrometer, which basically separates chemical compounds based on their mass. I now have a list of over 35,000 compounds that are found in my seaweeds, which is a lot! We are still trying to figure out an easy method to identify the compounds since the mass spec just gave us their masses, not their names, but a quick analysis on the compounds indicated that there were some differences in the types of compounds based on seaweed division (brown vs. red). Dr. Baker's lab also provided me with one more antarctic red seaweed, so the first thing I did when I got back to Charleston was run one final feeding assay and do my character trait assays on that seaweed. Since the holidays, I have completed all my trait assays and am in the final stages of generating DNA sequences from all my seaweeds. In the next few weeks I will be generating a molecular tree for my seaweeds (essentially a family tree) and will be working on trying to analyze and understand what all my data says!
I am actually in Florida right now, working with Dr. Bill Baker's lab at the University of South Florida. Dr. Baker and his students study the chemistry of marine organisms, such as seaweed, and much of their work is based on organisms from Antarctica. One of his PhD student, Jackie Fries, is helping me do some chemical analysis on my seaweed samples so we can determine the different chemical compounds in them. Ultimately, once we know the types of chemical compounds the seaweeds are producing, we can look for patterns to see if the chemistry is related to the seaweed type and/or where the seaweeds are originally from!
The past few weeks sure have flown by! I have been hard at work, continuing with my feeding assays (today marks seaweed 45!) while also trying to get back into the molecular side of things. I do still need to finish up my trait assays on the last couple of seaweeds before I have my final results, but trying to generate DNA sequences is currently at the top of my priority list. As of the last time I did molecular work, I had successfully isolated and amplified DNA from 26 seaweeds. So I re-extracted DNA from the 9 seaweeds that didn’t work last time and then also extracted DNA from the 17 seaweeds that were sent to us this summer. I then attempted to amplify the DNA in those seaweeds using PCR and all but 8 seaweeds worked (we can tell success based on gel electrophoresis). For the seaweeds that still were not successfully amplifying, I began troubleshooting the PCR reaction, first by increasing the amount of DNA in the PCR and then by diluting the amount of DNA in the PCR. I tried increasing the amount of DNA in case the DNA extracted was a very low concentration, in which case an increase in amount added to the PCR reactions would allow for better amplification. I also tried diluting the DNA in my brown seaweeds that didn’t work because brown seaweeds are known to have properties that can inhibit amplification if the DNA is too concentrated. Increasing the DNA amount led to successful PCR in 3 of the 5 reds while diluting the DNA led to success with the 3 brown seaweeds. Now there are just 2 seaweeds left to try re-extracting and amplifying again which I will try to do this week.
While we have had success with our PCR, we still need to see if it produces good DNA sequences. Before sending all of our PCR products for sequencing, we decided to send a subsample of 8 seaweeds for sequencing. I am aiming to get that done this week as well, so fingers crossed that we get good sequences back and then we can send the rest of our seaweeds for sequencing. The goal again is to get DNA sequences for all of our seaweeds so we can then construct a phylogenetic tree (a tree based on related ancestry- so think family tree). This tree will be used subsequently to look at the effect of phylogeny on the various seaweed traits and the overall palatability of those seaweeds to herbivores.
I have been hard at work the past few weeks trying to complete the character trait assays on the new seaweeds from Fiji and California. So far I have finished determining the protein concentrations, ash-free dry mass (which relates to organic content), and percent calcium carbonate in the seaweeds. We are seeing some latitudinal patterns (with the polar seaweeds being more nutritious)! We are also seeing differences between red (Rhodophyta) and brown (Phaeophyta) seaweeds, although I am still working through the analysis for confirmation on everything. Hopefully, I will be able to get you some figures (graphs and tables) soon!
I have also been continuing on with my feeding assays and am more than halfway through. We just got some more emerald crabs (Mithraculus sculptus) into the lab so we can do multiple feeding assays at once which should speed things up quite a bit. Moving forward, I am still going to be doing the feeding assays and am getting back into the molecular side of things trying to extract DNA from all my new seaweeds this week.
The past couple of weeks I have continued working on feeding assays with the crabs and urchins. We received some more Fijian seaweeds courtesy of our collaborator Mark Hay and we also received a few more temperate seaweeds thanks to Nicole who is now in CA. Our total number of seaweeds is now at 50 which is pretty exciting! I am about halfway through the feeding assays on the crabs and urchins and will begin working with the amphipods soon. I also will be starting trait assays on the new seaweeds sometime in the next week so stayed tuned for more on that!
This past week consisted of massive amounts of DNA extractions and PCRs!
I have been working with undergraduates, Sarah Shainker and Connon Thomas.
Sarah Shainker continued optimizing her restriction enzyme digest (see Sarah's research page for more information about restriction enzymes!) and now has the method nailed down. She started processing Gracilaria vermiculophylla samples from the native range in Japan, Korea and China and samples from our trip to the DelMarVa peninsula. So far, results are quite interesting with very clear patterns differentiating native and non-native ranges! Stay tuned for more about her research!
Connon Thomas continued his G. vermiculophylla growth experiments manipulating salinity for several South Carolina populations. He also got his feet wet doing high-throughput (high throughput means 96 or more samples processed at once as opposed to 5 or 6 samples) microsatellite genotyping on all his algal individuals.
I spent this past week doing PCRs with Sarah and Connon plus continuing with the samples we collected this summer along the DelMarVa peninsula and in South Carolina. Samples are now being run on the capillary sequencer (see the equipment page to learn more about the capillary sequencer). This week also saw installment 7 of my monthly phenology sampling of the DNR mudflat here at Fort Johnson. Though we found 1 male, all other individual G. vermiculophylla fragments were sporophytes or vegetative (meaning they were not yet reproductive).
I have been hard at work helping our high school student Hannah work through traits on Gracilaria. She is looking at how the amount of organic material, protein content, surface area to volume ratio, and palatability (how yummy it is) varies between males, females, and sporophytes. Males and Females are haploid so they only have one set of genetic material (DNA, chromosomes, etc) while sporophytes are diploid meaning they have two sets of genetic material--one from mom and one from dad.
I am continuing to do feeding assays with the urchins and crabs. I also spent some time this week collecting more amphipods (and one local type of isopod). I ran a choice assay with three different amphipod species and the isopod to see which animals would behave best for our experiment. While all of the animals behaved and consumed food from the feeding strips, we ultimately decided to continue on with Ampithoe valida since it is the most abundant species around Grice Marine Lab.
Courtney, Meredith, and I also collected some Diopatra from the mudflat and brought them into the lab in order to run an experiment with the Gracilaria chemistry we extracted last week. We are currently keeping an eye on the worms to make sure that they are happy and eating before running the experiment.
"An undergrad, Sarah Shainker, who is in the lab for the summer on a HHMI fellowship, and I just returned from a 9 day, 2500 mile and 25 site collection trip in VA, MD and DE. We are now processing all the seaweeds we collected, meaning we are beginning to extract DNA and then Sarah and I will be embarking on a joint effort. Sarah will be sequencing a single gene marker called cox 1 as well as doing some restriction enzyme work (basically chopping up the DNA into smaller pieces in which some samples get chopped one way and other samples get chopped another way). I will be tackling the genotyping of all the specimens with microsatellite markers (the same markers used in forensics and paternity cases in humans). My immediate goals will be to round up all the worldwide samples we have to finish off their genotyping and then get cracking on the new samples!
Next week another undergrad, Connon Thomas, and I will be headed as far south as Beaufort and as far north as Myrtle Beach to sample more Gracilaria for his Research Experience for Undergraduate summer project!"
"I am currently in the process of conducting choice feeding assays on the urchins and crabs. So far I have tested four temperate seaweeds, one antarctic seaweed, and one tropical seaweed. I have a total of 35 seaweeds in house and one of our collaborators (Dr. Mark Hay at Georgia Tech) is going to be sending us approximately 10 more Fijian seaweeds soon which is exciting!
I also spent some time this past week learning how to distinguish between amphipod species so I can collect enough of one kind to do feeding assays with them as well.
Additionally, I have been helping Hannah (our high school student) learn how to determine ash-free dry mass and protein content in Gracilaria vermiculophylla as part of her summer project."
Nicole has finished up her master's degree, meaning her research at the Sotka Lab has come to an end. She is moving on to her next scientific journey and we wish her the best of luck!
"I am setting up my experiment again. So, I have been going out into the field and collecting Gracilaria and then bringing it back to the lab to removing all the epiphytes (plants) and epifauna (animals) that are on it. Then I am breaking it into equal weight pieces and stringing it on to ropes so that I can redeploy the algae and not lose it!"
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Patriots Point Naval and Maritime Museum
40 Patriots Point Rd.
Mt. Pleasant, SC 29464
40 Patriots Point Rd.
Mt. Pleasant, SC 29464