Drafts

The end goal of this entire unit with dogs was to infer the genotype of the dogs at different genes. This was done by sequencing either the entire genome or, more practically, specific sequences containing desired single nucleotide polymorphisms (SNPs). There are many methods by which sequencing can be accomplished, but the most common method is Sanger sequencing. Sanger sequencing is run by amplifying desired DNA sequences in a solution containing regular deoxynucleotides (dNTPs) and a minimal amount of dideoxynucleotides (ddNTPs). These ddNTPs are structurally similar to typical nucleotides, but lack an OH group on the 3’ carbon that prevents the growth of the chain. As a result, sequences terminate at different lengths depending on the how early the ddNTP is added. The differently sized products of the amplification are then run through an agarose gel where they separate into bands. The distribution of the bands can then be used to read the sequence of the DNA. This method, while effective, can require lots of reagents and gels if more than a small sequence is being analyzed

The first step in establishing mutant lines is the induction of mutations in wildtype zebrafish. There are a number of ways to create mutations. A highly effective method is CRISPR/Cas9. It involves the injection of guide RNAs, Cas9 protein and stop oligos into embryos at the single-cell stage. CRISPR/Cas9 gives a high level of specificity to mutation induction. A wide variety of mutations are induced; some can be innocous point mutations and others can be insertions and deletions of varying lengths. This injected generation of fish is referred to as G₀. The injected adult fish that develop are mosaic organisms, meaning that different populations of cells have different genotypes (i.e. carry different mutations). To clean this up and have organisms with uniform genotypes, the G₀ adult zebrafish have to be outcrossed with wildtype fish. Ideally, the next generation of zebrafish (F₁) laid should have heterozygotes carrying one wild-type allele and one mutant allele, along with homozygotes carrying only wildtype alleles. Owing to the unpredictability of the process, all the embryos obtained from a cross could be wildtype. This might be an indication that the induced mutation did not go germline. To determine the alleles being carried by the progeny, a process called genotyping must be performed. That will be the discussion for the next segment. 

This is the write up for my microbial growth experiment.

Expected Results:

To do a viable count a serial dilution was done using E. coli. The original broth containing E. coli was diluted 8 times. It was expected that as the broth was more diluted less cells would grow on each subsequent plate. To test cell density E. coli was inoculated in 4 tubes of TSB and kept at different temperatures, 27, 37, 45, and 55 degrees celsius. The most optimal temperature for E. coli growth was expected to be 37 degrees celsius, this is because E. coli are adapted to live within the digestive tracts of humans and humans maintain a constant body temperature of 37 degrees celsius. The way cell density was measured was with a spectrophotometer. It was expected that the E. coli being incubated at 37 degrees celsius would have the biggest change in cell density. Additionally it was expected that E. coli would have the lowest cell density at 55 degrees celsius, this is because it is too hot for the cells to live at this temperature.

Observed Results:

The10-1and 10-2dilution plates produced lawns and the colonies were too numerous to count (TNTC). The 10-3dilution plate had 62 colonies on it, giving a density of 6.2*105CFU/mL. The 10-4dilution plate had less than 30 colonies which is too few to count. The 10-5, 10-6, 10-7, and 10-8dilution plates had no colony growth. The cell density over time experiment yielded expected results. The E. coli had most increased cell density at 37 degrees celsius and the least increase in density at 55 degrees celsius. The k value (number of generations per time period) and g value (generation time) were calculated for E. coli at all 4 tested temperatures. At 27 degrees celsius the E. coli had a k value of 0.013 and a g value of 75. At 37 degrees celsius the E. coli had a k value of 0.02 and a g value of 48.825. At 45 degrees celsius the E. coli had a k value of 0.012 and a g value of 82.748. At 55 degrees celsius the E. coli had a k value of 0.001 and a g value of 763.85.

The fact that the replicator observed a similar interaction, different from that which was originally observed, shows that this interspecific interaction is rather common. The tree photographed in the replicated figure has a narrower trunk than that in the original figure. This difference is likely due to insufficient detail given in the initial methods, as to the exact location of the tree observed. On the lawn between the Morrill Science Center and the University Club, there are several trees, so it is easy for a replicator to choose the wrong tree. Hence, in subsequent procedures, numerically quantifiable directions may enhance accuracy. The difference in arrangements can be attributed to the lack of arrangement specifications in the initial methods description. The absence of letter boxes in the replicated figure is because the methods description followed by the replicator did not include instructions about lettering. This should be included in subsequent method descriptions to enable more compliance with the original figure. The methods description also did not mention anything about spacing the panels in the figure; as such, the replicator had room to make assumptions. To curtail this, subsequent methods description must specify details about spacing. The initial methods followed by the replicator did not include adequate description of how to depict magnification, hence the absence of it in the replicate. More detail must be provided in future methods to enable proper execution.

Eutherians are another subclass of class Mammalia. The word eutheria comes from the Greek for "true" (Eu) and "wild beast" (Theria). Eutherians are unlike metatherians (marsupials) as they have a true placenta. The oldest eutherian fossil is the Eomaia found in China and dates back to the Cretaceous period. It is though to be small and insectovorous. Mammalian speciation expanded in two periods; 80-90 million years ago and 60-65 million years ago. For the 80-90 million years period the theory for speciation then is the super continent Pangea began to split apart and as the climate change so do niches, leading to some exctinction that would allow small mammals to fill new ones and rise. 60-65 million years ago was the exctinction of the dinosaurs allowing for more environments, niches, and resourses for the early mammals to use. Eutherians all share some distinct characteristics. Eutherians follow a primitive dental formula with slight variation based on species: Both the upper and lower jaw have 3 incisors, 1 canine, 4 premolars, and 3 molars, with a premolar that is clearly different from the molar. Females have a chorloallantoic placenta, vagina cervic uterus and ovaries again with variation based on species and males have a penis bone or baculum. 

In order to study the DNA of any organism, the genome must first be extracted for use. DNA extraction involves three crucial steps: tissue and cell disruption, preservation of the DNA, and clearance of extraneous cell components, including carbohydrates, proteins, and lipids. Once DNA has been purified, it is valuable to analyze the DNA sample in order to determine the yield and purity of the extraction product. Often times when DNA is extracted, RNA also remains in the contents since it is a nucleic acid similar to DNA; however samples can be treated with RNase, an enzyme that degrades RNA. DNA analysis can be carried out through gel electrophoresis, and for more information, it can also be spectrophotometrically analyzed. In spectrophotometry, the absorbance of light traveling through a sample is measured to determine the concentration and purity of the contents.

In this lab, we extracted the genomic DNA of Brachypodium distachyon. We then quantified the DNA using gel electrophoresis and absorbance spectrophotometry in order to verify the purification of the DNA, comparing samples treated and untreated with RNase A.

There are two categories of enzymes based on kinetic features, Michaelis Menten enzymes and allosteric enzymes. Michaelis Menten enzymes will display a hyperbolic curve when initial rate is plotted against the concentration of the substrate. They also have kinetic parameters that are often studied. The Km is the subtrate concentration when the rate is half the maximum rate. This value indicates how quickly an enzyme reaches max activity, and it can be considered a measure of affinity for a particular substrate. On the other hand, Kcat is the turnover number that tells how quickly an enzyme catalyzes a reaction after it bind substrates. It is determined in saturating conditions of an enzyme, and it is particular to specific substrates. The higher the Kcat, the faster the enzyme changes substrate to product once bound. These two value are then compared to determine the specificity constant, Kcat/Km. This is the measure of an enzyme's overall catalytic efficiency. It takes into account both subtrate binding and speed of product formation, so even if an enzyme's Kcat is low, if its Km is low, its catalytic efficiency can still be decent. These kinetic parameters provide information about an enzyme's function and can be used to compare various enzymes. 

          The Methods Project was an activity assigned in the Writing in Biology class during the Spring 2019 semester at the University of Massachusetts Amherst. The project aim was to produce a multi-panel scientific figure of an interspecific interaction and a set of methods for the reproduction of said scientific figure. The interaction displayed in my figure was between a fiddle-leaf ficus and a leafy vine plant which grows up its trunk. I needed to control for the day of the week and time of day due to Durfee Conservatory, which houses the aforementioned species, only being open during certain times, as well as the location of the photographer, orientation of the camera, and framing of the subject, the editing program used, and the the size, color, and placement of all of the components of the figure. I observed and documented the differences between the original and replicate figures and speculated as to reasons why these differences occurred. These differences could be categorized in five ways: as variations in color, framing, placement, size, and style of the components of each of the figures. The factors which likely contributed to the differences I observed between the figures were the lighting in the greenhouse, the position of the photographer, as well as the height and angle of the camera, unfamiliarity with the tools by which the components of the figures were sized and arranged, and lack of specificity in my methods. Through these activities, I learned about the necessity of clear communication in scientific writing to both convey the findings of a paper as well as allow for replicability. 

The first classification of taxonomy was proposed by Francis Willoughby and John Ray and Linnaeus used that same model of classification to begin his research on taxonomy. His model was strictly based on how to order species into groups but it had nothing to do with evolutionary relationships. Birds were mainly grouped into the aquatic or terrestrial habitats but not until after Darwin published his work of evolution that scientists started using his idea of bird’s classification, behavior, and morphology.

One possible area of therapeutic intervention suggested by McAllister and Weinberg would be inhibition of the intercellular signaling that ultimately leads to metastasis.  Small molecule inhibitors or neutralizing antibodies are what McAllister and Weinberg propose as options.  The authors mention that instigation into the clinical use of haematopoietic growth factors such as G-CSF, which is known to recruit pro-tumorigenic bone marrow cells, is ongoing.  Thus far, the clinical justification for this type of treatment seems dubious due to the pro-tumorigenic role of G-CSF.  Anti-angiogenic therapies have been improved and continue to be researched.