Superlab

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!