The aim of this experiment was to use wild type and mutant strains of Chromobacterium violaceum to explore N-acyl-homoserine lactone (AHL) based quorum sensing in Gram negative bacteria. AHLs are signal molecules produced by Gram negative rods. They regulate antibiotic synthesis, expression of virulence genes, biofilm formation, and several other cellular activities. Two genes are responsible for AHL mediated gene regulation. One encodes a transcriptional regulatory protein (R gene), and the other encodes the enzyme AHL synthase (I gene). The presence and proper functioning of these two genes is essential for the target genes to be transcribed. AHL synthase produces AHL molecules, which are classified by their side chain length and molecular structure. AHL synthases differ between each genus of bacteria, and produce AHL molecules that are slightly different from each other. Most regulator proteins that bind AHL molecules are specific for a certain AHL structure, but some can bind more than one type of AHL. This can create the phenomenon of cross-communication between different species of bacteria. This experiment was done to explore quorum sensing in Gram negative bacteria and determine if different species of bacteria could communicate with C. violaceum.
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The aim of this experiment was to explore the wide variety of organisms found in biofilms that form on toothbrushes. My lab partner’s used toothbrush was the source of these bacteria, and was sacrificed for the purposes of this experiment. The toothbrush head was cut off and placed into sterile saline to create a fluid suspension. An inoculum from the toothbrush-saline suspension was streaked onto TSA, SBA, MacConkey agar (MAC), Mitis-Salivarius agar, and CDC anaerobe blood agar plates. After incubating the plates, it was expected that each one would contain several different colonies that would allow the bacteria growing on the toothbrush to be directly observed.
The results of this experiment were in accordance with my expected results. On the slide culture of P. aeruginosa, a thick, slimy, green goo had grown and covered the entire slide and surrounding glass dish. When I attempted to remove the glass slide from the dish, long thick strands of biofilm formed between the slide and the glass dish. It was like when you take a bite of cheese pizza and the cheese forms a long, gooey strand between your mouth and the slice. When I put the P. aeruginosa slide under the microscope at 400x, I could see long, thin, greenish grey strands of biofilm going in all different directions. It was clear from this slide that P. aeruginosa formed a biofilm that contained lots of EPS. At 1000x the strands were not as defined, but rod shaped bacteria with thin EPS and biofilm filaments in the extracellular space were clearly visible. This showed that P. aeruginosa is an excellent biofilm former, and can form biofilms in an artificial environment in the lab.
The aim of this experiment was to observe biofilms microscopically by growing them on a slide using a process called slide culture. Using a technique called flow through Gram stain, the biofilms on the slides were kept wet to maintain their complex arrangement. Keeping biofilms wet is essential to preserving their structure, and allows them to be seen under a microscope. The inoculum for these slide cultures was obtained from two different sources. The first slide culture sample was taken from a pure culture of Pseudomonas aeruginosa. This Gram negative, rod shaped bacteria is an excellent at forming biofilms, which contribute to its virulence in humans and animals. It was expected that P. aeruginosa would produce a thick, slimy, dense biofilm on the slide culture. It was also expected that extracellular polymer substance (EPS) would be able to be seen under the microscope.
When a water soluble substance, like Vitamin C, is present in the body at higher than threshold
plasma levels, it will be eliminated from the body through urination. The level of the substance
in the body must reach the renal threshold for this to begin to happen. In the case given in this
example, we are dealing with vitamin C which is a water soluble protein that can’t be stored in
the body. If you were to intake too much vitamin C into your body at a given time, then it will
remain in your blood stream until it is eliminated via the kidneys and urinated out of the body.
Taking the vitamins in smaller quantities in timed out intervals will allow all of the vitamins to
be absorbed into the body because the renal threshold will not be reached.
Overall objective: to observe the phototactic behavior of the common cellar spider through the effects of LED light on web formation
Specific Aim 1. Observe how LED lights hanging at three different distances from the top of three separate Ziploc container effect cellar spider web formation. The first container’s LED will be touching the top of the container, the second container’s LED in the middle of the container, the third located one inch from the bottom of the container.
Phototaxis is the directional movement of an organism towards or away from a light source. This behaviour has been observed in a wide range of organisms, from unicellular bacteria to complex multicellular organisms. Phototaxis can be positive or negative. Positively phototaxic organisms move towards the light source, while negatively phototaxic organisms move away from the light source. Each organism has its own specific biological cause for a phototactic response. The phototactic response of the common cellar spider in response to artificial light is a mechanism that is poorly understood. In this proposal, we aim to observe the phototactic behaviour of the common cellar spider using light emitting diodes (LEDs) arranged in six different controlled environments, and determine if the artificial light source has any effect on the spider's web building behaviours. To do this, medium, square, Ziploc Tupperware containers will be modified with LED lights and house one cellar spider for seven days. We propose six modifications of the containers to test the spiders phototactic behaviour in various environments:(1)Different lengths of LED light insertion in the container (2)Different colors of LED light displayed (3)Different spider species used (4)Different brightness of light displayed (5)Different time exposures of light and (6)No light present in the chamber. Phototactic behavior will be quantified by measuring the distance between the LED light and the spider web (if present) and its diameter. The data from all six environments will be compiled and analyzed using an Excel spreadsheet. Understanding the effects LED light has on phototactic responses of spiders, specifically web formation, will have profound implications on our understanding of animal behavior and pave the way for future research projects.
Before 2002, the dogma surrounding rod and cone cells was that they were the only cells with photosensitive properties in the retina of the eye. It was also known that light entrainment and the circadian cycles are tuned by environmental influences. Scientists knew that exposure to different light cycles can reset the circadian clock, but did not know how this worked. Humans and mice that lacked rods and cones could still reset their circadian rhythms. They hypothesized that there must be another mechanism in the eye besides rod and cone photoreceptors to allow for this photosensitivity. Their question was: Could melanopsin be a photo pigment protein that allows retinal ganglion cells (RGC’s) to be light sensitive?
The results from the E. faecalis were also not in accordance with the expected results. Very little growth was observed when E. faecalis was inoculated on GYE side of the GYE/water Petri dish. I expected to see more growth on the GYE side of this dish, but only a few small colonies were present. This could be due to the inoculation process only depositing a few bacteria onto the paper strip. Another possibility is that GYE does not contain the necessary nutrients for E. faecalis to grow. On the control plate with GYE on both sides, E. faecalis did not grow at all, and no colonies were observed. This showed that GYE may not be the preferred media of E. faecalis to form colonies. This could also be due to a flaw in the inoculation process.
This experiment showed that S. marcescens has motility. When this bacterium used up all of the recourses at the point of inoculation, it was able to move towards the side of the Petri dish with more nutrients. This organism was unable to grow sufficiently enough on the water agar to move towards the GYE. The tests with E. faecalis did not provide definitive evidence that this organism is non-motile. This organism did not grow well on either media, and its ability to move could not be observed. Using a different media could allow for the motility of E. faecalis to be observed more clearly. In conclusion, this chemotaxis experiment provided evidence of motility in S. marcescens, but was inconclusive in determining the motility of E. faecalis.