The Role Of Spna As A Cell Wall

The C. elegans were washed several times once a week for several weeks. After the final wash for each week, a small amount of C. elegans was pipetted to the middle of each plate (3 experimental plates and 1 control plate). After all the passages for the C. elegans were completed, the three avoidance assay plates were set up. (A small amount of S. marcerens was pipetted on one side of each assay plate while a small

Cin1 exists as Cin1-S (short-form) and Cin1-L (long-form) isoforms as a result of alternative splicing of intron #6. Cin1-S differs from Cin1-L by lacking the C-terminal RhoGEF (DH)-PH domain. We previously generated a CIN1-S mutant strain by replacing the C-terminus with an NAT marker gene. Significantly, we have found that Cin1-S confers a survival advantage over WT in the mouse brain via co-infection. Further investigation will include constructing a mutant expressing the long isoform Cin1-L. We have generated a partial CIN1-L point-mutation allele linked to G418 resistance, but have yet to obtain the Cin1-L mutant through the conventional gene replacement method. Recently, we have developed a one-step CRISPR-CAS9 technique for mutagenesis in Cn. In this aim, we will generate the Cin1-L mutant, characterize its function, and perform co-infection assays. Moreover, we will perform dual RNA-Seq to profile whether there is differential gene expression in infection by Cin1-S and Cin1-L in order to address the mechanism associated with Cin1-S CNS survival

Restriction Enzyme Digestion – The experiment was begun after putting on gloves to avoid any chemical contact with the skin. Four microtest tubes were obtained, and each of them was labeled to contain the different enzymes or suspect DNA. Two of the microtest tubes were used for suspect one and the two different restriction enzymes, while two other microtest tubes were labeled for suspect two and the two restriction enzymes. After labeling the tubes, the contents that were at the bottom were taken out by slightly tapping them. Then to begin setting up the enzyme reactions, a micropipette was used to obtain 10 μL of the reaction buffer which was added to each of the four test tubes. The buffer is important because it carries the electrical current from the power supply in the gel. After the reaction buffer was in each, the microtest tubes were individually filled with their specific enzymes and DNA, shown in summary through Table 1.1 below. The restriction enzymes are used to cleave the DNA at specific

N2 control worms (M=6.67, SD=1.53) had statistically significant more tail thrashes than unc-54 control worms (M=0.67, SD=0.58), p=.002. There were also statistically significant more tail thrashes in N2 sugar worms (M=8.33, SD=2.08) than unc-54 sugar worms (M=2.00, SD=1.00), p=.001. There was no statistically significant difference observed between N2 stevia (M=3.00, SD=1.73) and unc-54 stevia (M = 1, SD = 0.00).

cross-links the cell wall to make it strong.[1] Making the cell wall is easily one of the most

The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.

revealed the existence of abundant amount of microsporidia in the bloodstream of H.axyridis under light microscope and again confirmed using electron microscope. Once microsporidia enters the bloodstream, it undergoes RNA transcription inside the cell. But in H.axyridis it is in inactive state due to absence of gene transcription factor and doesn’t cause any harm in them perhaps it has established immunity towards them. This forms a shield for H.axyridis eggs as well as larvae but when ingested by native ladybird species i.e. Coccinella beetles leads to death. Amplification of microsporidia shows that its RNA matches closely with Nosema thomsoni. This is present in all H.axyridis by vertical

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

Although the two resistant and five sensitive colonies’ DNA sequence and their response to the AHL Sensitivity Assay were as predicted, this information alone does not give us a measure of their long-term response to exposure to the killer. We thus asked, “Have the resistant colonies evolved to be completely resistant to the killer and have the sensitive colonies (even though they had no detectable change in the protein E gene sequence) evolved to respond to the killer in a different manner?” To answer these questions, we tested the two resistant prey colonies (PreyR1 and PreyR2) containing transposon insertions and the two sensitive colonies (PreyS1 and PreyS2; previously exposed to the killer) against the killer (Table S1). As expected, the killer (dotted at the centre) had no apparent effect on either PreyR1 or PreyR2 growth (No kill

The purple colored plate was given as the wild culture with no mutations and observed by a group of three persons. Observations of the adults included that they were the fastest moving and they also were the ones to move around the most. The embryos were clearly seen in the body and they often trampled over the other worms. There were fewer embryos in the culture and they were immobile. They were clear in the middle with black outline and sometimes hard to see. The smaller larva clustered into piles. However, when the plate was dropped and tapped, the worms scattered and moved faster than they were before. Even tickling the worm produced a reaction of them moving even when they weren’t before. There were about 10% embryos, and 18% of adults, L1, L2, L3, and L4 worms. For the green colored plate, the group found the mutation to be a roller. It was observed that the worms were in a circle or horseshoe shape and 100% of adults seem to be in this position and not moving very often like the wildtype. Even when dropped, the worms did not move very quickly, if at all. There was also fewer small larva. The red colored plate was observed to be the Dpy-10 mutation or also known as dumpy. These worms were slow moving, short, and fat. Some of them didn’t move at all, and tapping did not increase the movement at all. The worms seemed to be at a length of .05 mm. There was also less large larva

Worm-star assay: Staged adult animals will be washed in M9 buffer three times to remove residual bacteria. Approximately 10,000 animals will then be incubated for three hours at room temperature in 5 ml M9 buffer (without OP50 bacteria) in 100 mm petri dishes tilted at a slight angle to concentrate animals in a single area of the plate. The number of animals in worm-star aggregations, clusters of two or more animals entangled at their tails, will be quantified by visual inspection using a dissecting microscope. (Schultz 2014)

In this investigation, 2-aminophenol(AP), 4-chlorobenzaldehyde(CB), the metal salts CuCl2.2H2O (99.00%), Pd(OAc)2 (99.99%), and AgNO3 (99.50%) were utilized and purchased from Sigma–Aldrich Chemise (Germany). The important reagents for DNA interaction studies, Calf-thymus DNA (CT-DNA), 2-amino-2-hydroxyl methyl- propane -1,3- diol (Tris) and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma – Aldrich. CT-DNA dissolved in Tris-HC buffer (ph7.2). Tris-HCl buffer solution was prepared using deionized water and was used to control the pH of the reaction system. TAE Buffer (Tris-acetate-EDTA) (50X), Top Vision Agarose, Ethidium Bromide Solution (10 mg/mL), 6X DNA Loading Dye and 100 bp DNA Ladder are utilized

Specifically, we will find if Cin1 orchestrates the only endocytic pathway and how it affects host-parasite interaction through regulating exRNA transport and mouse CNS survival. We propose three specific aims: Aim 1 is to determine if Cin1 mediates the sole endocytic pathway in Cn through identification and characterization of pathway components. Aim 2 is to examine the role of Cin1 in exRNA export through continued annotation and analysis of RNA-Seq data. Aim 3 is to further explore the CNS survival advantage of Cin1-S by generating the Cin1-L strain using one-step CRISPR-Cas9 mutagenesis method we recently developed and testing it through co-infection.

In all areas of biology, it is easy to see that structure is related to function. This statement holds true in microbiology as well, the study of microorganisms, including bacteria. One characterizing feature of bacteria is the cell wall, which can generally (although not in all situations) be categorized into one of two categories: either Gram positive or Gram negative. Gram positive bacteria’s cell walls are composed of a large peptidoglycan layer (up to 90% of their cell wall). Within this large peptidoglycan layer, one can find techoic acids, which contribute to the maintenance of cell wall structure, and lipotechoic acids, which attach to membrane lipids. Gram positive bacteria that act as pathogens can also potentially release exotoxins, which can have very dangerous effects on humans. Gram negative bacteria, on the other hand, have a very small layer of peptidoglycan in their cell wall, which is surrounded by an outer membrane. Within the outer membrane, one can find the lipopolysaccharide layer, which is one of the most distinguishing factors of Gram-negative bacteria. It is important to note that Gram negative bacteria fail to possess techoic

Cn is an encapsulated basidiomycetous fungus that infects both immunocompromised and healthy individuals to cause life-threatening meningoencephalitis [14]. Cn produces virulence factors including melanin, the polysaccharide capsule, and extracellular ureases and phospholipases [15-17]. Previous studies showed that secretory transport is important for virulence [18] and that Cn has proteins with conserved specific functions in exocytosis, such as Sav1, Sec6, and Sec14 [19-21]. Previously, we identified cryptococcal intersectin 1 (Cin1) as a novel endocytic adaptor protein that is important not only in intracellular transport but also in growth and virulence [22]. Cin1 contains multiple domains including one EH domain, one CC region, an actin monomer-binding WH2 domain, two SH3 motifs, and a RhoGEF-PH domain. Also, the Cin1 domain structure shares more similarity with mammalian ITSN1 than Sc Pan1 (Fig. 1). Further studies suggested that Cin1 functions upstream of Wsp1, a GTPase-binding domain (GBD) containing Wiskott–Aldrich Syndrome protein (WASp) homolog and a Rho family GTPase, Cdc42, to regulate actin