Thursday, December 19, 2013

Yeast Lab 12/19

Purpose: The purpose of this lab was to demonstrate cell communication through different stages of the mating of yeast.
INTRODUCTION: Cells communicate through the release of chemical signals which illicit varying responses in the unicellular colonies. In this lab there are 2 types of yeast (a) and (alpha) they each secrete a mating (a) and (alpha) factor respectively. When the (a) type yeast cell receives the (alpha) factor and the (alpha) type yeast cell receives the (a) factor a change in the cytoskeleton of each yeast cell type undergoes a change. Each cell cytoskeleton elongates because of the opposite cell factor binding to a receptor on its cell membrane that caused a specific signal transduction pathway. The cytoskeleton elongating forms a shmoo, each shmoo veers towards one another and finally meet to form xxa zygote.
Cells communicate in order to relay information and thus illicit a cellular response. One way cells communicate is through g-protein coupled receptors. A signal stimulates the receptor to undergo a transduction pathway. During this transduction period, the signal molecule activates the receptor to displace GDP to GTP. This causes the g protein to activate, thus relaying the information it got from the receptor to a neighboring inactive enzyme. The active protein activates the enzyme, thus evoking a cellular response.
Methods:  We obtained alpha, a, and alpha/a mix yeast with broth in three different test tubes.
We made 3 different wet mounts that contained the alpha, a, and alpha/a cultures on each slide. To avoid contamination, we used three different pipettes when creating the wet mounts.
We placed these wet mounts under a light microscope. The zoom was calibrated, as it was difficult to focus on the mass of yeast cells. First we looked at the cultures at a X10 magnification then under a X40 magnification.
Then we looked for fields in which the yeast cells were possible to count. We chose 3 fields per wet mount. We took readings at intervals of time 0, 30 minutes, 24 hours, and a final reading at 48 hours. We recorded the amount of haploid, shmoo, zygote, asci, and budding cells there were per field.

Discussion: We isolated the two different groups into a isolated and alpha isolated. The two isolated cells did not show much  communication. In the 24 hour period, there was a decrease of alpha and an increase of A cells, but at the 48 hour mark there was an increase of A cells and a decrease of alpha cells. It seems as though both cells had
opposite living conditions under which they survive the best. Also, the budding of both cells might have undergone some fluxes due to various concentrations in different parts of the wet mount we saw. For example, the presence of air bubbles in the wet mount. The air bubbles pushed away the cells, making them compact and harder to count.
The mixed group showed many stages of cellular communication. We saw haploids, shmoo, zygotes, spores, and ascus. This, unlike the isolated colonies, showed a much higher rate and greater sophistication of cellular  communication. The formation of all of these was due to the sexual reproduction between alpha type and a type yeast cells.
The most particular formation was the pear-shaped shmoo cell. It gets its pear shaped from its ability to recieve a ligand (signal) through a g-protein coupled receptor. (to see how a g-protein works, see the introduction). The formation of shmoos would be an example of local signaling as the cell changes shape from within. Because the yeast cells don’t have the motor skills to move, it grows to a potential mate. The g protein coupled receptor sends that signal and tells the cell’s nucleus to grow towards where the signaling molecule concentration is the highest. Thusly, the cell will have a bigger chance to mate. This explains the pear shape of the shmoo as it grows towards a potential mate.

As you can see here is the Alpha yeast. This is the isolated apha culture that was allowed to reproduce asexually. You can see the rise and the fall of both the haploid and the budding haploid population
Below you have the Alpha Yeasts and A yeasts as isolated cultures. You can see the rise and fall of both Haploids and budding haploids over the 4 periods of time.

Based on our findings, we concluded that yeast cells communicate via direct contact as well as pheromones. Since the yeast cell does not have the ability to move on its own, the easiest way it can mate is by fusing with a close, neighboring cell. Pheromones come into play when the cells are more spread out. Signaling molecules (ligands) are ejected from the cell, thus motivating the alpha/a cells to grow towards where the signals are of greatest concentration. This produces shmoos. Once in close proximity, mating and sporulation occurs. Proximity is an important factor as to where shmoos will appear first in a petri dish with both alpha and a type yeast. Since it will be most efficient to send a ligand over a shorter distance, shmoos will appear first in the area of least distance between alpha and a type yeast. In a hypothetical situation, if we are given a petri dish with 4 distinct regions marked in the petri dish and 3 of the circles contain alpha type yeast and one circle contains a type yeast, presence of schmoos will first be detected in the alpha circle closest to the a type circle.

Conclusion: We concluded that the most cellular communication happened when the a culture was mixed with the alpha culture. The zygotes, shmoos, haploid cells, and asci proved that communication between alpha and A yeast cells needs more signals to carry out more complex responses. On the other hand, we have the separated a and alpha cells which, on their own, did communicate but only formed haploids and budding haploids. This asexual reproduction allowed for the yeast to multiply though not as fast like the mixed cells.

References: Lab

Thursday, December 5, 2013

Chromatography and Photosynthesis/Light Reactions Lab (12/5)


Photosynthesis Lab
    The purpose of this lab was to test how boiled and unboiled chloroplasts and the presence of light affect the transmission and absorption of light in a solution of phosphate buffer, distilled water, DPIP, and chloroplasts.

Chromatography Lab
    The purpose of the paper chromatography lab was to calculate the Rf values of the different pigments in spinach. The purpose of this lab was to separate the different chlorophyll that exists in spinach leaves. This would separate the individual chlorophyll according to their size. Also, we used a color coder to reveal which specific chlorophyll we were actually seeing.


Photosynthesis Lab
    Set up an incubation area with a light, water flask and test tube rack, respectively. Keep the containers containing the unboiled and boiled chloroplasts solutions on ice. Label cuvettes 1,2,3,4,5, respectively. Cover cuvette 2 with tin foil. Label test tubes 1,2,3,4,5, respectively. Add 20 drops of phosphate buffer to all test tubes. Add 80 drops of H2O to test tube 1. Add 60 drops of H2O to test tubes 2,3 and 4. Add 63 drops f H2O to test tube 5. Now add 40 drops of DPIP to all test tubes with the exception of test tube 1. Transfer all solutions in the test tubes to their corresponding cuvettes, fill cuvettes 3/4 full. Now with respect to the spectrophotometer adjust the amplifier control knob until the meter reads 0% transmittence. Add 3 drops of unboiled chloroplasts to cuvette 1 and place it in the sample holder, adjust knob to read 100% transmittence by turning knob to the red light. You will now add a drop of unboiled chloroplast solution to cuvette 2, and immediately take a reading using spectrophotometer, removing tin foil to do so. After the reading place the cuvette in the test tube rack in the incubation area covering it with its tin foil. Let it sit for 5 minutes and then take another reading. Don't forget to recalibrate the spectrophotometer before every reading. You will take reading in intervals of 5 minutes up until 15 minutes. In respect to cuvette 3 also add a drop of unboiled chloroplasts. Cuvette 4 add a drop of boiled chloroplasts. Cuvette 5 will be used as is. Follow the same guidelines for readings and place cuvettes in incubation area in same intervals.

Setting up the individual cuvettes

Here is the device in which we placed the cuvettes in. It shone red light through the sample and then recorded it. 

This is our set up. Light is being separated via water and enters the samples. 

Chromatography Lab
    Obtain a long test tube. Add 1 cm of the provided solvent. Cut a piece of paper in a manner that it is long enough to reach the solvent but short enough to stopper the test tube. The paper will also be cut with a point, this end will touch the solvent. Draw a line 1.5 cm above the point. Place spinach leaf on top of pencil line. Rub the leaf with the edge of a coin. Repeat several times using a different part of the spinach leaf. Place the pigmented paper in the test tube now with the point barely touching the solvent and stopper the tube tightly. When solvent is 1 cm from the top mark the front. Remove the paper from the tube and mark the bottom of each pigment. Measure the distance the pigment migrated from the origin pigment line.

Extracting the pigments 

Isolating the paper with the pigment. Note the color. 

After about 10 minutes or so, there is already noticeable color separation of pigment. 

Measuring how far the pigment traveled. Cork was used to keep the paper from bending. 

Photosynthesis Lab
    The light in the flask will absorb most of the infrared radiation from the light and transmit most of the visible radiation. Wavelengths of light within the visible light spectrum power photosynthesis. Cuvette 2 will be covered with tin foil to prevent light from coming in it is a control group. The test tubes will provide room in which substances can become diluted. There is more H2O added to test tube 1 and 5 because test tube 1 will be used for calibration, and the extra H2O in test tube 5 replaces the chloroplasts. Cuvette 1 will be used to recalibrate between readings it demonstrates the measurement of a 0 and 100% transmittence, this is the scale on which the other cuvettes will be measured by. Recalibration between readings will reset the spectrophotometer.
Chromatography Lab
    In order to calculate the Rf values for the different pigments we observed, we used the equatiom for Rf values. Rf = (distance of pigment migration) / (distance solvent migrated). 


Photosynthesis Lab
    The phosphate buffer is used in the expirement in attempt to slow the light reactions, because in reality the reactions take place extremely fast. H2O in the experiment provides the electrons for the light reactions to take place in the chloroplast. DPIP is our form of NADP in the chloroplast which will be reduced by accepting electrons that are passed down the electron transport chain. So the light will hit the cuvettes and power photosynthesis. Each cuvette will contain chloroplast to a different degree. Depending on the contents of each cuvette and its conditions whether covered by tin foil or with boiled chloroplasts, different results will be obtained. In general H2O will donate electrons which through a series of steps including the passing through PS1, ETC, and PS2 in the chloroplasts; DPIP will be the last acceptor and there will be a change in color in the cuvettes from blue to colorless as DPIP is used. 
    As you can see in our graph the highest rate of absorption happened when the chloroplasts were unboiled and were exposed to light. This makes sense because the chloroplasts were not altered or boiled, and they were exposed to light which provided the partial fuel it needed to  carry out its reaction. 
    DPIP, as stated before, is an electron acceptor that takes place of NADPH. In our first attempt at this experiment, we used too much DPiP and not enough chloroplasts to yield good results. The fast acting DPiP already finished reacting with the chloroplasts, thus finishing the reaction by the time we got to our 2nd run. As a result, our data was unreliable past the second and sometimes third run.  In order to improve this, we decided to increase the amount of chloroplast while keeping the amount of DPiP the same. This would allow the reaction to go at a more gradual rate as DPiP has more chloroplasts to react with.  This made our data more gradual and reliable. We also calibrated our device in between every run to ensure that our data was as accurate as possible. 

Chromatography Lab
    For the paper chromatography lab, we were able to get 3 distinct bands of pigment. The first pigment we observed had an olive green color. An olive green colored pigment corresponds to chlorophyll b. The second pigment we were able to distinguish had a bright green color; bright green colored pigment indicates chlorophyll a. The last pigment we were able to see had a yellow color, indicating the pigment xanthophyll. It is worth noting that chlorophyll b and chlorophyll a were located closer to the marked line and did not travel that far up the filter paper. This is most likely due to the fact that the oxygen and nitrogen in chlorophyll make it cling to the paper more, preventing it from traveling farther up. Another interesting observation we made was that the xanthophyll pigment was located farther down the paper; the explanation we had for this phenomenon is that xanthophyll is not very soluble in the solvent we used.
    The solvent moves up the paper mimicked capillary action. This this is a result of the attraction of the solvent molecules to the paper (adhesion), and the attraction of the solvent molecules to one another (cohesion). As the solvent moves up it carries with it the pigments dissolved in it. The pigments travel different distances up the paper because they are soluble to the solvent unequally. Also each pigment attracts to the paper fibers differently. A pigment can bond to the paper fibers through H-bonds. In this manner certain pigments can near the solvent front more than other pigments when they are more soluble in the solvent and when they don't form H-bonds with the paper fiber. On the other hand other pigments will travel less and not near the solvent front when they aren't very soluble in the solvent and create strong H-bonds with the paper fiber.

Graphs & Charts:

Photosynthesis Lab

In this graph we have the rate of transmittence in percentage with the amount of time it took to transmit that amount. 
Run 1: Unboiled with light
Run 2: Unboiled chloroplasts with no light
Run 3: Boiled Chloroplasts with Light
Run 4: No chloroplast with light 

Here we have the absorption rate of the different runs. 

Run 1: Unboiled with light
Run 2: Unboiled chloroplasts with no light
Run 3: Boiled Chloroplasts with Light
Run 4: No chloroplast with light 

Chromatography Lab

Calculated Rf for each color that we saw. 


Photosynthesis Lab 
    In this lab we concluded that chloroplasts will react at the highest rate when they’re exposed to light and unboiled (not denatured). In contrast, the slower rate of light reactions occurred when the chloroplast is denatured and/or in the dar. That way the chloroplasts have no way of carrying out a light reaction. 

Chromatography Lab
    In this lab we found out the chlorophyll presence in the cells of a spinach plant. Using the chromatography paper, we concluded that the most prevailing cholorphylls were xanthophyll, cholorphyll a and chlorophyll b. In that order, from most present to least, the chlorophyll traveled up the chromatography paper separating the layers.