Hi everyone! So unfortunately, our third attempt at PCR didn’t work… and neither did our fourth attempt. We started our fifth attempt this morning (and we’re going to end up running over 90 tubes this attempt!) so let’s hope we get something this time. After our third round of PCR didn’t work, we hypothesized that we might not have enough genomic Anopheles DNA, so we decided to use another group’s DNA that we knew worked. At the same time, we decided to raise the extension times and play with the annealing temperatures to try to get a band on our gels. To be able to move on to the next step of this process, ligation, we would have needed to see bands by last Monday. Since we didn’t see anything, we knew we wouldn’t be able to continue on and finish everything by the end of the quarter, so we decided to just focus on troubleshooting our PCR. We decided that since we’re just going to focus on PCR we would also troubleshoot Canoe, in an effort to get SOMETHING. Unfortunately, at this point we still haven’t gotten any bands on our gels, which makes us think there may be something wrong with the primers that we’re using to extract the genes. This weekend we’re going to try running a variety of semi-nested PCR reactions to test to see if any of the primers are defective. Had our PCR worked, as most groups’ did, we would have ligated our PCR product into plasmid vectors, and then transformed these vectors into competent E.Coli bacteria, similar to what we did in Superlab last quarter with our bacterial DNA. After transformation, we would have prepared mini-preps of the DNA from bacterial cultures, sent the samples away to be sequenced, and then analyzed the sequences to determine if they were what we were expecting to find. If our PCR this weekend works, we may still be able to ligate and transform, but unfortunately we’re so close to the end of the quarter we won’t have time to send it away for sequencing and get it back before the end of next week, which is disappointing, but that’s science! This coming Friday, we’re going to give in-class presentations about what we did this quarter and our results, so we anticipate our presentation having a LOT of PCR!
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Happy December! The past couple weeks have been really stressful, what with Thanksgiving break and midterms for almost all of my classes, but I have some time to post again, now that the majority of my work is done (for now). A lot has been going on in Superlab lately. The day before Thanksgiving break, we set up our second round of PCR, which was nested PCR in an effort to extract only our gene. This past Wednesday, we ran all of our samples from both the first and second rounds of PCR on a gel (12 samples total) and imaged it to see if we got bands in the correct places. Unfortunately, our gel looked pretty strange. Heartless amplified a ton of DNA, so much that the band pattern looked like a DNA ladder (what’s used to determine the length of amplified DNA fragments), which looked nice but really wasn’t what we wanted to see. On the other hand, Canoe didn’t amplify anything, so we ended up striking out twice. Luckily, this isn’t unusual. Only one group in our lab section actually got a band at the correct size, so our situation isn’t too rare. We decided that for time’s sake we’re going to focus on Heartless, rather than trying to clone two different genes, since we were starting to go in so many different directions. We hypothesized that the strange amplification pattern observed was due to incorrect primer dilutions, so we re-made our stock primer solutions and re-ran the first and second rounds of PCR, this time using different temperatures and semi-nested PCR, which is similar to nested PCR but uses a different combination of primers. We ran those samples on a gel this morning, and, drum roll please… got nothing. We think something may have gone wrong with the first round of PCR such that no DNA was amplified, which is disappointing, but gives us a starting point for troubleshooting. I’m going back into lab in a little while to re-make our first round PCR reactions, and hope that the third time’s the charm! We’ll be able to run the second round of PCR tomorrow morning, and run all the samples on gels tomorrow night, so that if we get any product (fingers crossed!!) we’ll be able to start the next step of the process—ligation—on Monday afternoon. If we don’t get bands a third time, we’ll be able to troubleshoot more, and hopefully get something by Wednesday. At this point in the quarter, with only two weeks left before finals, we’re really getting down to the wire, so we really have to hope we get some usable DNA this week!
Hey everyone! This was a busy week in Superlab. On Monday we had our first journal club of the quarter, where we discussed the paper “Control of Drosophila Gastrulation by Aplical Localization of Adherens Junctions and RhoGEF2” by Kolsch, et al. I’ll post the link below if you’re interested in reading more about it. Unlike last quarter, we have three journal clubs this quarter, so rather than each lab section presenting on a particular day, we divided into groups based on which paper we wanted to present (I’m presenting tomorrow on actin-myosin networks in constriction during gastrulation), and groups of two or three people present the various figures from the paper. During lab days this week, we performed genomic DNA extractions and set up the first round of PCR with our first set of primers to begin gene amplification. On Wednesday we were given small dried mosquitoes, and our task was to grind them up and extract DNA from them using a series of reagents we prepared in lab last week. On Friday, we measured the concentration of our genomic DNA preparations using a little instrument called a Nanodrop that used absorbance readings from our samples to measure concentration of DNA. Unfortunately, our preparation had a relatively low concentration of DNA (luckily, most people in the class had low concentrations, so it wasn’t that abnormal), but we’re going to go ahead and use it, so hopefully it works! If not we should have time to go back and re-perform the DNA extraction if necessary. Along with measuring DNA concentration, we set up the first round of PCR on Friday to amplify specified sequences of DNA using the first set of primers we designed in the first week of lab. If all goes well, the week after Thanksgiving we should be able to run a gel on both our first and second rounds of PCR (we’ll be doing the second round in lab this week), and hopefully we’ll see product! If not, we’ll have to go back and troubleshoot, but we’ll cross that bridge when we reach it. Until then, we have journal clubs and PCR to work on! Have a great Thanksgiving everyone!
Journal club article 1: Control of Drosophila Gastrulation by Apical Localization of Adherens Junctions and RhoGEF2
Journal club article 2: Pulsed contractions of an actin–myosin network drive apical constriction
Hi everyone! Yesterday we had our second day in lab, after learning about PCR theory and techniques in lecture on Monday. PCR (polymerase chain reaction) is a technique used to amplify pieces of DNA that we’ll be using throughout the semester as we try to isolate gastrulation genes. We used this technique a lot last semester to when we were sequencing bacterial DNA, but this quarter it’s up to us to make our reagents, and trouble shoot everything that can go wrong with this procedure. In lab yesterday we got to choose which gastrulation genes we want to study and isolate throughout the quarter. I’m working in a group of three, so we decided to choose two genes to work on simultaneously. We chose the two genes Heartless (fibroblast growth factor receptor 1) and Canoe. Heartless is a gene that’s involved later in the gastrulation pathway than we’ve studied so far. Its main function is differentiation of cells of different lineages, and cell spreading of the mesoderm (the middle germ cell layer in an embryo that eventually forms connective tissue). It’s also involved in development of the central nervous system, the morphogenetic furrow and cells surrounding the hindgut in Drosophila embryos. It’s nicknamed Heartless because in its absence organisms don’t develop a heart. Canoe is involved in cell-cell adhesions and junctions between cells. After choosing a gene yesterday, our goal was to research its function and sequence, and from the sequence choose a region that we want to clone. Genes are composed of both introns and exons. An exon is the portion of the sequence that is later transcribed into RNA, whereas the introns are removed by RNA splicing, and are not present in the final RNA product. We chose the largest exons in our sequences to clone, and then used online programs to create primers to these exons. These primers are made of 18-20 base pairs that are complementary to the beginning and end of the exon, and they hybridize to that chosen part of the genomic DNA sequence, allowing it to be amplified through PCR. The primer sequences we chose were sent off to be created by an external company, and in a couple of days we’ll get our primers back and can start isolating genes! In the meantime, we have to prepare for DNA extraction so that we can actually use the primers, so tomorrow we’ll begin preparing solutions for genomic DNA extraction. I’ll keep you updated on how that goes!
I’m back! The second quarter of Superlab started on Wednesday, and we’ve already hit the ground running with our new projects. Every quarter throughout the year we switch professors and projects, so this quarter we’re working with Professors Rachel Hoang and Judy Owen. Our project for the quarter involves studying gastrulation of the mosquito Anopheles gambiae. Gastrulation is a process during development of an embryo in which the embryo is transformed from a ball of cells into various layers of tissue. Mechanisms underlying gastrulation are related to other biological events such as the development of vertebrates and the progression of particular cancers, making the study of how this process is controlled particularly important. Previous research on this topic has focused on understanding the array of genes that control gastrulation in Drosophila melanogaster, a type of fly that’s considered a model organism. Our project for the quarter involves investigating the genes that control gastrulation in another type of invertebrate, the mosquito. Each pair of students is going to spend the quarter isolating and cloning a particular gene involved in gastrulation from the Anopheles gambiae mosquito. This will involve performing a number of different techniques, including DNA isolation, PCR, ligation and transformation, mini-preps and sequencing—many of the same techniques we used last quarter to sequence bacteria from various plants. On Monday we get to pick the gene that we’ll spend the quarter analyzing and start preparing for DNA extraction, so I’ll keep you updated!
Since hurricane Sandy threw off our schedule for the week, we held the introduction lecture on Wednesday, and visualized embryos on Friday to identify different stages of gastrulation. Each pair was given a plate with Drosophila embryos, and the goal was to identify embryos that were in different stages of development, and then observe them over a period of time as they gastrulated. The embryos all looked like tiny plankton, and identifying different stages of development was difficult due to minute differences in color and shape that separated them. Luckily, I was able to isolate six different embryos that were about to start gastrulation at stage 5, and watch as they proceeded through the four distinctive stages of gastrulation to stage 9. The class as a whole took some really good pictures of the process, so as those come in I’ll post them here (stay tuned). We also looked at fixed mosquito embryos, which were very different from the fly embryos. They looked sort of like brown bananas, and it was impossible to see the same structures that were visible in the fly embryos, indicating that gastrulation is very different between the two. It was really fun to watch the fly embryos develop, and I think this is going to be a really exciting quarter!
Hello again! Quarter 1 is now officially over, and Superlab wrapped up last Thursday and Friday with poster presentations. Last time I posted, I was on my way home for fall break and didn’t have sequence data yet, so a lot has happened since then. Three weeks ago, we sent 11 different samples off for sequencing, which came from 6 different original bacterial colonies. We ended up getting usable sequence data back for all 6 colonies, which was exciting, since we’d been having such bad luck up to that point. As it turned out, the six colonies we were able to sequence corresponded to the following bacterial species: sample 3 was Enterobacter cloacae, sample 10 was Sphingomonas aerolata, sample 12 was Pseudomonas brenneri, sample 13 was also Sphingomonas aerolata, sample 15 was also Pseudomonas brenneri, and sample 16 was Stenotrophomonas maltophilia (see phylogenetic tree at the bottom for species diversity). As it turned out, these six colonies corresponded to four different species of bacteria. We went on to analyze and compare sequence data from the two other groups in our larger group. One group (KUOUAK samples on the tree) analyzed data from sassafras, a type of dicot, and the other group (DDJJ) analyzed data from a conifer. Unfortunately, none of the sequences from the second bamboo sample came back positive, so we were unable to compare diversity between the two bamboo samples. Of note, many of the samples from both KUOUAK and DDJJ were phylogenetically similar to our bamboo species, indicating that there was a lot of overlap between species among the different types of plants.
The whole point of this quarter of superlab was to analyze the phyllospheric diversity of plants on Haverford’s campus. Our original hypothesis was that plants in similar macroenvironments, ie, plants that were located close to each other in similar areas hosted similar bacterial populations. Although we have too few samples in our data to really confirm or refute our original hypothesis, based on the data we were able to tentatively conclude that macroenvironment does contribute to the diversity of bacteria on different plants. However, outliers, such as the Curtobacterium ammoniigenes found on the conifer, lend support to the idea that bacterial populations are somewhat dependent on host species. Local host environment of the leaves may play some part in determining what kind of bacteria can survive. For example, most of our bacterial species are typically found in harsh environments, indicating that they may be able to live on the nutrient-poor surface of bamboo leaves. The Curtobacterium found on the conifer typically lives in acidic environments, so it may find the surface of a conifer needle more inviting than a bamboo leaf. Of note, according to primary literature, almost all of our bacterial samples are typically found in aqueous environments, and it was really humid and rainy the week we originally collected samples, indicating that the water might have attracted the bacteria, or could have washed them onto neighboring plants. Also of note, almost all of our bacteria were gram-negative bacilli, indicating that gram-positive bacteria may have been resistant to PCR or other techniques used throughout the quarter. In conclusion, while we can neither confirm nor refute our hypothesis, preliminary data lends support to the idea that both macroenvironment and microenvironment of the plant leaves has a role in determining phyllospheric diversity.
If we had another seven weeks, there are a couple of things that we would want to try with this experiment. First of all, we would re-run our culture-independent analysis so that we would be able to sequence a wider-range of bacteria. We would also try different methods to try to get more gram-positive samples, such as boiling samples before PCR at a higher temperature. We would also want to re-run analysis of bamboo leaves from other parts of the plant, which would give us a better idea of whether macro or microenvironment played a larger role in the determination of bacterial diversity. From there, we would want to analyze and compare the phyllospheres of bamboo plants in other locations, both on and off campus. We would also want to compare diversity on different parts of the plant during different seasons and weather conditions.
Overall, this was a great quarter. I came into the year knowing little about bacteria, and hardly anything about plants, and I feel like I really learned a lot about both in the past seven weeks, and I can’t wait to take quarter classes in the spring to learn more! Not only did I learn a lot about the concepts behind what we were doing, but I became much more comfortable with lab techniques pertaining to both, which will definitely be useful in the future. Beyond concepts and lab techniques, I got more experience with analyzing data, making posters and presenting our findings to people both in and outside of the scientific community, which I think is a really important skill to have, and why superlab is such a fantastic course!
The second quarter of superlab was supposed to start today, but due to Hurricane Sandy it’ll start Wednesday. I’m not sure if there’ll be a blog for the quarter or not, so stay tuned, and for now enjoy pictures of our phyllogenetic tree and poster!
Hello again! I’m currently sitting in an airplane on my way home for fall break! I’ve been trying to update my lab notebook, but my pen just exploded everywhere and the fasten seatbelt sign is on so I can’t clean it up, so I’ll update you on what’s been going on the past few days. We’ve almost made it through the quarter at this point (just one week left) and our projects are quickly wrapping up. On Wednesday, after doing a restriction enzyme digest overnight, we ran all of our samples on a gel, and we got 11 samples that showed positive bands! These 11 samples came from six different colonies, which means we’re bound to get some kind of data, so it was a huge relief since we haven’t been having such great luck lately. We sent all the samples away for sequencing, and theoretically we should get data back today, so as soon as I land that’s the first thing I’m checking (after I see my family and everything of course)! Once we get sequence data back, we’ll be able to use the online database BLAST to match our sequence to those of similar organisms, to try to determine the species of our individual bacteria. From there, we’ll be able to create a phyllogenetic tree comparing our samples to the samples of the other people in our group, so we can finally begin to answer our original question: does phyllosphere depend on local environment or plant species? In the meantime while we wait to get our sequence data back, my partner and I have been working on creating our poster, and it looks really good so far! It needs to be done so we can print it by next Wednesday, so after we print it I’ll post a picture of the poster with some of the conclusions we came to. Next Friday on the last day of class we’ll hold a poster session so that everyone in the class can present their posters to each other and our professors. It’s exciting to see that all of our hard work over the past six weeks is finally paying off, and I’ll keep you updated on what we discover!
Hello again! The past couple of days have been spent trying to wrap up aspects of our project and get to the point where we can send off our samples for sequencing. Since we had to, unfortunately, drop the culture independent portion of our project, we’ve just been focusing on our culture-dependent samples, and luckily we have a lot of them. Friday night my partner and I went into lab to do a pre-plasmid purification preparation on our samples, Saturday we went in to do a Qiagen miniprep on them, and yesterday we started a restriction enzyme digest. At this point, we’re doing the same thing to our culture-dependent samples that we did with our culture-independent samples, so fingers crossed we have more luck this time! Today in lab we’re going to run a gel to see if everything worked, and if so we’ll actually be able to send something away for sequencing, which would be great since our poster is due just two weeks from today!
On Monday we had our second journal club, but this time my lab section presented the figures to the rest of the class. As a class we voted on which article we wanted to read, and we chose a really interesting article about carrying capacity called “Variation in Local Carrying Capacity and the Individual Fate of Bacterial Colonizers in the Phyllosphere” (I’ll include the link below if you’re interested in reading it). Carrying capacity is generally defined as the maximum number of bacteria a leaf can support. Through the use of computer models and green fluorescent protein labeled bacteria, the authors challenged the commonly accepted definition of carrying capacity (namely that the capacity was the same across the surface of a leaf) and asserted that the leaf is actually made up of different sites of high, medium and low carrying capacity, which in combination sum to give the total carrying capacity of the leaf. They demonstrate that there are a large number of sites on the leaf that have either a medium or a low local carrying capacity, and a small number of sites that have a high carrying capacity (meaning they have a lot of nutrients and bacteria that land there can divide many times). Thinking about the carrying capacity of a leaf in terms of specific sites on the leaf rather than as an average value for the leaf as a whole gives us another tool with which to analyze our own samples. Although we can’t use GFP-labeled bacteria or computer models, analyzing the plant anatomy can help us think about where on the leaf there might be sites that are better equipped to handle a large number of bacteria, and where bacteria might have trouble growing. Using our initial plates from the beginning of the semester, we can come up with the carrying capacity for our own leaves, to determine whether or not bamboo is a good habitat for bacteria. I’ll keep you posted on how that goes, and whether or not we can send samples away for sequencing!
Hi everyone! So, unfortunately, we got some disappointing news today. As it turned out, the gel that we thought looked so good last night didn’t actually look as good as we thought. Although there were bands present in both sample lanes, they weren’t in the right place (so they weren’t the right length), which means the ligation didn’t go well. Since we only have two weeks left, at this point we have to scrap the culture-independent portion of our experiment, which is disappointing since we put so much effort into it, but that’s science! Many of our culture-dependent transformations seem to be growing well, so later tonight we’ll go in and do pre-plasmid purification preparation (try saying that five times fast) on them, so that we can do a Qiagen miniprep and run a restriction enzyme digest on them tomorrow afternoon.
Since we’re done with the culture-independent project and our culture-dependent plates are still incubating, today we focused on staining bamboo stem and leaf sections. The sections turned out really well, so that was exciting! We used three different stains today: Toluidine Blue O (TBO) which is a metachromatic stain, meaning that you can apply this single stain to one sample and it’ll stain multiple features of the sample different colors. It stains pectins (sugars) purple, lignins (found in wood) blue green and cuticle dark blue. We also stained with Alcian Blue, which stains cellulose (in cell walls) bright blue. The final stain we used today was Safranin, which stains lipids and waxes reddish orange, and lignin dark red. The final stain we’ll use later next week is Phloroglucinol, which stains xylem bright red, but you need to use it with hydrochloric acid, so we won’t use it until later. We got to view the sections we did stain with a microscope in the basement that allows us to take high-quality photos, which I’ll include in this post—they turned out really well, gave us a lot of information about the anatomy of a bamboo plant, and will look good on our poster!
Good news! The last two days in lab have been really successful, and we’re finally getting to the point where we’re close to results. On Tuesday evening, I went into lab and prepared our two culture-independent samples for Qiagen column miniprep, which we did in lab yesterday. This is a multi-step process that purifies our plasmid DNA. After purifying our samples, we ran a restriction enzyme digest. The enzymes cut our DNA in specific places, so tonight I ran a gel to estimate the size of the cut pieces and to make sure our bacterial DNA inserted correctly. Our gel looked really good, and the bands were in the correct place, so tomorrow we can send our two samples off for sequencing! I’m not sure how long it’ll take to sequence them, but hopefully by sometime next week we’ll get results and can start comparing our sequences to a database that will (hopefully) allow us to determine the species of bacteria present in our phyllosphere.
Yesterday, along with doing the Qiagen column miniprep, we re-ran a gel of our culture-dependent PCR products, because last time our negative control lane was contaminated. Luckily, when we re-ran the gel our negative control lane came back negative, so we were able to proceed with this portion of the project. What was really exciting was that all but one of our samples came back positive for bacterial DNA, so, assuming everything works from here on out, we’re going to have a lot of culture-dependent data! At this point, we need to run basically the same procedures on our culture-dependent samples as we did on the culture-independent samples. Yesterday we performed a ligation on our samples, and tonight we went into lab and transformed them. Part of the transformation process involves spreading two agar plates per sample, so I ended up spreading 34 plates! Our samples are currently taking up half the incubator, but by tomorrow, if everything goes well, we’ll be able to do pre-plasmid purification prep on our samples, run a Qiagen miniprep and restriction enzyme digest on them on Saturday, and run a gel on Sunday. If everything looks good, we’ll be able to send our samples out to be sequenced on Monday! I have my fingers crossed that everything goes well in the next few days, especially since we’re getting so close to the end of the quarter (2 weeks!) that it’s time to start thinking about what we’re going to put on our poster that we’ll be presenting the last week of class.
When we had some down time yesterday we collected more bamboo leaves (with stems) for future staining. Something interesting I learned about bamboo is that it has flowers! Professor Wilson showed us that at the base of the leaf, where it connects to the stem, are small hair-like structures, which are specifically designed for wind pollination. We also learned that, although bamboo isn’t full of pectin (sugars), it might actually be a good habitat for bacteria, which would explain why we have so many different samples. Even though the leaves don’t have a lot of nutrient sources, they also don’t produce anything that’s deleterious to bacteria, unlike daffodils, which apparently produce a substance that’s toxic to most types of bacteria! I included some pictures of our bamboo flowers and one of our pre-plasmid purification preparation plates from Tuesday.