In this paper (Bansal 2017), a several methods were used to carry out experiments in their study. Planarian culture was the first method to be enacted, which detailed how they were maintained prior to the start of the experiments when they were killed. After killing, they performed whole-mount in situ hybridization and double fluoresence in situ hybridization. Some specimens were then immunostained "whole-mount", as opposed to smaller sections of them. Then, they also did take smaller sectioned samples by "embedding and cryosectioning", before proceeding to immunostain those as well. Also following the in situ hybridization, BrdU labeling and immunofluoresence staining was done. In regards to imaging these samples, both with confocal microscopy and transmission electrion microscopy were utilized. Then, for further sample analysis, they performed polysome profilling, followed by qRT-PCR, mRNA purification and transcriptome sequencing, all of which confirmed the hypothesized significance of their protein of interest.
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In this paper, a several methods were used to carry out experiments in their study. Planarian culture was the first method to be enacted, which detailed how they were maintained prior to the start of the experiments when they were killed. After killing, they performed whole-mount in situ hybridization and double fluoresence in situ hybridization. Some specimens were then immunostained "whole-mount", as opposed to smaller sections of them. Then they also did take smaller sectioned samples by "embedding and cryosectioning", before proceeding to immunostain those as well. Also following the in situ hybridization, BrdU labeling and immunofluoresence staining was done. Following this, the samples were imaged both with confocal microscopy and transmission electrion microscopy. Then, for further analysis, they performed polysome profilling, followed by qRT-PCR, mRNA purification and transcriptome sequencing.
You have a protein you wish to identify the location of. You make two GFP fusion contructs, one at the N-terminus, one at the C-terminus. When the fluoresence is visualized, the one with the N-terminus tag seems to be localized to "dot-like structures", while the C-terminus tag looks to to be in a "network-like structure" in the cell. Describe a method in which to confirm the localization of the N-terminus unknown protein tag.
A common method that can be used to determine the localization of unknown protein is co-localization with marker-fusion proteins. In this method, a GFP fusion contruct is made with your protein of interest, GFP being a green fluoresent "tag" onto the protein sequence, and this contruct can then be stably transformed into a plant genome. At the same time, another fusion construct can be made, which is the "marker-fusion protein" construct, meaning the protein's function and location are known, then for a tag, a different fluoresent color such as red or yellow is used. The marker-fusion protein construct is then also stably transformed into a plant. The resulting two homozygous plants are then crossed, and "co-localization" can now be checked for in the crossed progeny. Specifically, this is done by observing the progeny plant cells with fluoresent microscopy; if an overlay of the two colors is observed, the localization of the unknown protein is then confirmed.
The goal of this project was to strengthen scientific analytical skills through practising replication and distinguishing between observations and inferences. The aim was to create a multi-panel scientific figure, detailing how it was produced, and then passing on these instructions to another in hopes that they would then create the same if not a very similar figure. The similarities and differences between the created figures were then inferred and observed upon completion. Approaching this, I thought a tree would be a good organism that could be easily located, with features that would be easy to find and identify. I ended up choosing a European Beech tree, Fagus slyvatica, as it was pretty large and set apart from other trees surrounding it. I aimed to control the location of the specific tree, the different aspects of the tree that pictures would be taken of, as well as the angle at which the photos were taken.
Once I had fully compared the replicate figure to my own original one, I realized I was not entirely surprised with some of the major differences between the two. After the submission of my methods, I thought of several small details that I had left out that would have definitely contributed to more accurate replication, and did indeed result in the less accurate replication described above. When initially detailing my figure methods, I felt it was most important to specify exact location so that whoever followed them would definitely find the exact tree depicted in my figure and not a different one. However, I then failed to think of additional small details when actually taking the photos, details such as time of day, which is what lead to the overall noticeable difference in lighting between the two figures. I also did not specify the distance at which each photo was taken, resulting in the different depths in the figures.
From the two figures (Fig.1 and Fig.2), a number of observation differences could be seen. Upon initial glance, the figures are definitely not the same overall shape. Fig. 1 is a square, while Fig. 2 is not uniform, the different pictures within the figure stick out in a couple different places. There are no borders between the photos, and surrounding the picture in Fig. 2 as there are in Fig. 1. It was also noticed that the letters labeling the photos, while in the same general location, are not set into the specific upper left hand corners of the photos in Fig. 2 and are not the same color font, nor do they have the same background color as with Fig. 1. Going further into the details of the actual photos, another difference is how the arrows and text boxes appear in the two figures. In Fig. 1, the arrows are small, long, thin and placed within the photos of the figure. Meanwhile in Fig. 2, the arrows are the same color, but are larger, they point from outside the overall figure to the inside, and two of the total three arrows are not in the correct picture. In Fig. 1, the two arrows are placed inside picture B - both pointing to the bark, in Fig. 2, the two arrows come from the outside of the figure pointing onto the bark within picture C. Then, with regards to the text boxes, one can observe that boxes in Figure 1 are blackish gray in color, are translucent, have white colored font for the notes, and are positioned inside of pictures A and B at the opposite end of the arrowheads. In Fig. 2 however, these boxes are clear, with a white background from being placed outside the pictures instead of inside, having black font for the notes, and of course not in the same placement in relation to the pictures compared to Fig. 1.
The research paper, “Spatial Auxin Signaling Controls Leaf Flattening in Arabidopsis”, aims to gain more understanding of the mechanisms behind leaf flattening through observation of leaf development in Arabidopsis thaliana. A diagram is provided illustrating a leaf primordium sectioned off into different regions labeled with what gene is believed to be the most active for that region. The “adaxial” and “abaxial” sides are also labeled, simply referring to the regions which will either grow to face towards the plant stem (adaxial), or on the opposite side, growing away from the plant stem (abaxial). They then go on to explain that two genes in particular, WOX1 and PRS, are known to be significant in enabling leaf flattening from the middle domain, however it is not known specifically how these genes come to be expressed and that is what they intend to discover in their research. Based on previous studies, they believe that the nature of the two gene’s expression is similar to genes in the shoot apical meristem (SAM). In order to figure this out, they pursue multiple methods. First, they did some initial experiments with auxin and ARFs (auxin response factors) and saw overlap between auxin signaling in the marginal domain and expression of WOX1 and PRS. Then they performed a series of experiments following this all testing the relationship of one significant ARF, called MP, with WOX1 and PRS expression. They found MP to be a promoter of their expression in one experiment were they made a mutant MP, changing it from a promoter to a repressor and thus resulting in downregulation of WOX1 and PRS. In another experiment, they further confirmed this relationship by observing ectopic MP activity, with a another kind of MP mutant, and found that WOX gene expression was induced in the areas where the mutant MP was active. In their final testing with MP, they found it to be a direct up-regulator of WOX1 and PRS expression. In their final following experiments, they tested the other ARFs (2 , 3, and 4) relationship with WOX1 and PRS expression, and found that ARF2, ARF3 and ARF4 all suppress expression of the genes in the abaxial domain, also by direct binding like with MP. Thus, the nature of WOX1 and PRS expression is regulated both by direct binding to the MP promoter and direct binding to the ARF repressors.
In this paper, the collective researchers sought to find out more about how the different electron transfer pathways contribute to delta pH formation in the thylakoid membrane. In order for the production of ATP to occur as a result of photosynthesis, a proton gradient across the membrane needs to occur, and this comes about by way of linear electron flow (LEF) and cyclic electron flow (CEF). It is not known just how much either pathway contributes to the overall change in pH formation, so to figure this out they employed the use of 9-AA fluorescence quenching. They used the pH indicator, 9-Aminoacridine, and mixed it in with isolated chloroplasts to then monitor the “fluorescence intensity” of them, which was used to estimate the delta pH across the membrane. Under CEF, they looked at three mutants of two specified pathways under this; a PGR5 deficient mutant (pgr5), an NDH deficient mutant (crr4-2), and a PGR5 overexpression line (35s::PGR5#2). Using the method above, they illustrated the delta pH levels of each in graphical figures. In one experiment, they measured the pH of each mutant, in addition to wildtype (WT), at differing light intensities. This showed that the PGR5-dependent pathway has a contribution of around 25-30% because the deficient mutant had a noticeable reduction in delta pH from WT, meanwhile, the NDH-dependent pathway has a contribution of around 5%, with the deficient mutant showing little to no impact on delta pH. In a second experiment, they then determined the effect of light intensity on non-photochemical quenching induction in the leaves of WT and mutants. Similar patterns were observed where c224-2 (NDH-dependent) was closer to WT levels, meanwhile pgr5 was significantly reduced, suggesting that induction of non-photochemical quenching in wildtype is presumably because of PGR5-dependent pathway contribution to delta pH formation. This data also suggests that delta pH formation is most likely associated with non-photochemical quenching induction. The researchers then took their combined data and compared it to another recent study which estimated “the significance of CEF for generating proton motive force (PMF)…composed of delta pH and the membrane potential” which lead them to further conclusions. Together the studies indicate that linear electron flow primarily contributes to delta pH and PMF at lower light intensities, while PGR5-dependant cyclic electron flow then becomes more dominant under higher light intensity conditions.
Comparing orginal and replicate: In general terms the four pictures making up the figure are the same in each figure made. A is a picture of the lower trunk of a tree showing a large mushroom specimen. B is an overhead/arial view of the location of the tree with the mushroom on it, on campus, with a pushpin exactly where it is. C is a bar graph illustrating the distance from the "mushroom" tree's general location to the 3 closest bodies of water - the Campus Pond, Puffer's Pond and Lake Warner. D is again picture of the tree with the mushroom on it, but this time it is taken from several feet away to illustrate what the overall shape of tree looks like presumable so that it's easier to idenity which tree has the muchroom on it. Once again - each picture is the same general picture in each figure.
Contrasting original and replicate: While all four pictures are generally of the same thing, there are still noticeable differences. Picture A (which puts the mushroom on the tree in full view) differs mostly in that original was clearly taken during the day, and the replicate was taken in the evening with the help of flash from a camera. The angle at which the picture is taken is also slightly different but overall the way the photos are center is very similar, just the time of day is obviously different. Picture B is the photo that differed the least between the two figures, as it's in the same photo format, the only difference is the placement of the photo. The original has a more upward, zoomed in placement, leaving the pinpoint of the tree location more towards the bottom of the picture, while the replicate image is a bit more zoomed out and has location of the tree in the center of the image. Picture C also had similar images, minor differences in bar coloring for each body of water distance, and then the original just has the exact distances printed over each bar whereas the replicate does not. Picture D had the same differences as in picture A where the images were clearly taken at differing times during the day, but other wise the placement of the photo is almost exactly the same.
When I began thinking about places on campus containing interesting organisms, of course being fascinated by plants I thought of a myriad of locations because our campus is so filled with beautiful varieties of plant life. However, I did end up remembering a nice grouping of trees inhabiting an enclosed yard next to Durfee Conservatory while considering options, and . I made my way over to that area and entered the gated yard, which sits adjacent to the conservatory on the south side, from the back end that is closest to central campus. I then walked over to the labeled set of three gigantic trees; a gingko biloba, a european beech and a fernleaf beech. I explored around each one them, looking at their trunks, branches, leaves, how close their branches were to the ground, if there were any leaves on the ground, if there were any nuts on the ground, etc. I then decided to choose the tree whose trunk was the biggest of the three. I stepped back several feet in order to take a “portrait” photograph with my phone including as much of the tree as possible. I then walked back towards the tree and got much closer. I angled my phone up slightly, and took a picture of the upper portion where most of the branches were, while also including detail of the trunk. Finally, I went over to the left from the tree where I saw a branch from the tree hanging low enough to be within reach. I found and held in place a branch that was most accessible, and which also had a clear view of the leaf shape. I then took one final picture holding the branch in way that provided a good angle for seeing a leaf.