pGLO Lab

The purpose of this lab was to perform the procedure of genetic transformation.  In this procedure we will be transforming bacteria with a gene that codes for Green Fluorescent Protein (GFP), which as a result will glow green under a black light.

pGLO plasmid is a plasmid that is used in making genetically modified organisms. The plasmid glows green because of the many reporter genes. The gene for GFP, which is encoded by pGLO plasmid, can be switched on in transformed cells when some sort of energy source is added to the cell. Cells that are transcribed and remain white if they do not contain arabinose, which is like a food to the cell, and cells that appear to be a fluorescent green are cells that have the sugar added to them.

First we labeled two separate micro test tubes +pGLO and –pGL. Then we added 250ul of transformation solution to each tube, and then immediately placed then over ice. After that we scooped up a single colony of bacteria and mixed it into the solutions of the tubes labeled +pGLO and –pGLO. After we examine the pGLO DNA solution under the UV lamp, we mixed a new sterile loop in the the pGLO plasmid DNA stock tube. We scraped a loop full of plasmid DNA and mixed it with the +pGLO test tube and NOT the –pGLO test tube. After that we let the test tubes sit on ice for ten minutes. In the meantime, we labeled our four agar plates, LB/amp +pGLO,  LB/amp/ara +pGLO,  LB/amp –pGLO, LB –pGLO. After the ten minutes was up we kept out test tubes in the sponge holder and placed them in a hot bath for 50 seconds.  Immidietly, we out the test tubes back onto ice for another two minutes after the 50 second hot bath. After the two minutes we added 250ul of LB broth to each test tube to act as food to keep the bacteria alive and to help them recover from the various temperature shocks. After letting the bacteria incubate in room temperature for ten minutes, we placed 100ul of +pGLO bacteria into the LB/amp +pGLO and LB/amp/ara +pGLO dish and the –pGLO in the the LB/amp –pGLO and LBn-pGLO dish. We then evenly smeared the bacteria around the dish and then closed them and stacked them upside down and placed them in the incubator.

We put the CaC12 in the bacteria in order to allow DNA to enter.  By placing it in the bacteria, the positively charged CA2 of the CaC12 will cancel with the negative charge of DNA allowing it to pass through the cell membrane of the bacteria.  By keeping each test tube submerged in ice, the bacteria is able to maintain its shape while allowing the DNA to enter.  When we heated the tubes, we heat shocked the bacteria.  This makes the DNA enter the bacteria.  By heating it, we are expanding the area for the DNA to enter.   After putting the tubes back in ice, this allows the gaps to close, keeping the DNA inside.  This process has created a new pGLO plasmid.  The broth is then used as food for the bacteria and allows it to create new proteins that are amp resistant.  By making it amp resistant, the amp  can no longer destroy the bacteria.  The bacteria is able to create more of the pGLO plasmid which in part glows in the dark.  

After analyzing our data, we are sorry to report that none of our plates glowed in the dark.  This could in part be because we failed to wait a certain amount of time after adding the broth.  Also, perhaps we had not successfully heat shocked our tubes as we were unsure if our tubes had successfully touched the hot water. However, we can safely conclude that when 

E. coli glowing in the dark after put under a UV light (glowing E. coli credits of lab group 6)

Gel Electrophoresis Lab

The purpose of this experiment was to figure out the location and amount of cut marks that each restriction enzyme had compares to the next

The point of this lab is to determine who or what unidentified DNA belongs to. The three restriction enzymes, in this case are PstI, Hpal and SspI are set up on three different columns. Once the enzymes are loaded into the gels the enzymes undergo gel electrophoresis. This procedure makes the enzymes separate into their cuts. This allows a scientist to evaluate who or what the DNA belongs to based on the cut marks.

To begin this experiment we poured about 5mm of agarose solution into a casting tray. Then we scooped out a large bubble of debris and added to the side of the tray while it is still a liquid. Once the agarose has set, we placed the tray in the gel box so that the slots are at the negative end. Once the slots for the DNA are submerged completely, the DNA is ready to be loaded. We then carefully extracted small amounts of the DNA out of the tubes, and then steadily inserted them into the chambers of the gel. It is very important that the gel does not break at any time, for the experiment will be ruined. Once they are loaded, the electrophoresis box is closed and connected to electrical leads. After some time, due to the shocks from the voltage source, the DNA begins to move along the gel, splitting at certain points. After the DNA has split down the entire gel, we took it out and examined the DNA cuts and determine what the DNA belongs to.

After examining our gel, we noticed that the DNA had travelled to a different area than where we had initially put it.  The DNA of each moved toward the positive end of the gel.  This is because DNA is naturally negative due to the phosphate backbone so it wants to move opposite from the negative end of the gel and is attracted to the positive end.  Also, the distance travelled by each strand of DNA was different as well. This is in part due to the size of the strands.  Bigger strands of DNA tend to not move as much as the smaller ones due to the fact it is harder for something bigger to travel a long distance.  The bigger strands are unable to move through the gel as easily as the smaller ones; in a sense, they can't fit.  They were the ones closer to the wells or the initial positioning of the DNA.

Our experiment allows us to conclude about the effectiveness of gel electrophoresis when looking for a DNA match. Each band moves a different distance because restriction enzymes only cut at their specific protein recognition sites. 

The gels after the DNA had been added, inside the machine having electricity run through it

Finished gel after having been shocked

Strawberry DNA Extraction: How To

First we put the strawberry into a plastic bag with some of the 'DNA Extraction Liquid' and mashed it into a pulp.

Next, we put a coffee filter over a graduated cylinder and poured some of the pulp into the filter paper. We let that drip into the cylinder for a few minutes.

After we had enough liquid we transferred that into a test tube so that we could see it better. 

We then added some rubbing alcohol to the test tube and watched DNA extraction happen before our eyes. 

Once we saw some of the DNA, we used a little hook to take some if it out of the test tube. The DNA we took out was a sort of goo. 

Cell Communication

The purpose of this experiment was to calculate the percentage of yeast at each stage before and after a night of incubation.

This experiment is observing cell communication in yeast cells. Yeast cells are unicellular fungi that can reproduce sexually and asexually. For a yeast cell to reproduce sexually, the change their body shape into a gamete called a shmoo. When the a-type and alpha-type schmoo fuse together, the two nucleus’s to form  diploid nucleus with an a/alpha- genome. From there a zygote forms which then begins to divide into daughter cells. Yeast do require a time of incubation before they begin to divide, but once they begin to divide they continue at rapid rates until the area gets too populated, then the death phase takes over.

First we obtained agar plates and culture tubes in which we would grow and store the yeast. We labeled them alpha-type, a-type and mixed. We then scraped a small amount of each type of yeast, placed 2mL of sterile water onto a microscope slide then looked at each slide carefully. We observed and recorded approximately how many yeast cells we saw. After we were finished with that, we gave the yeast in the culture tubes some broth to last them overnight, then placed them in the incubator. The next morning we repeated the same procedure. We observed and recorded how many more yeast cells there were due to the yeast cells mating overnight. 

The amount of yeast increased because yeast reproduces asexually.  This means that it is able to reproduce without the help of a partner.  When yeast reproduces, it creates shmoos.  When shmoos of different yeasts touch, they combine creating more yeast. However, in order for them to touch they have a sort of attraction that pulls them together.  In the end, the amount of yeast  created was more than 10 times as great after a twenty four hour period.  When we first peered into the microscope, before the twenty four hour time had elapsed, we noticed that the mixed yeast already had connected with other cells more often than the isolated a or alpha types.  Some of the mixed had already connected with five other shmoos.

From our experiment we can conclude that reproduction of yeast cells can occur after spending a night in an incubator. Yeast cells are able to communicate with each other as long as there is fuel for the cells. Cell communication can happen within a cell or between two cells, this is represented by the ability of yeast to reproduce. We could have run into some errors in regards to which yeast cells are which as we forgot to label some of the pictures.

Mixed type yeast before twenty four hours had passed

a-type yeast before twenty four hours had passed

Alpha type yeast after twenty four hours had passed

Plant Pigments and Photosynthesis

4A: Plant Pigment Chromatography

In this experiment we used paper chromatography to measure the movement of pigment from plants. The mixture of solvent and pigment moves up the paper due to the attraction of solvent molecules to one another. In plants Beta carotene is the most commonly found carotene found in plants and attracted near the solvent because it has no hydrogen bonds with cellulose. The chlorophylls in plants are filled with oxygen and nitrogen and bind much tighter to the paper then the other pigments

In this experiment we wanted to use chromatography to separate plant pigments and isolate chloroplasts by using dye DPIP and then measure the rate of photosynthesis

First we got a 50mL graduated cylinder that had one cm of solvent and got a piece of filter paper that would be long enough to reach to solution. We then smashed a spinach leaf on top of the piece of filter paper with a coin to extract the pigment out. Once the pigment was on the paper we stuck it in the tube so the pigment was just above the solvent. Then we let the solvent be absorbed into the filter paper until it was about a cm from the top. Each time we noticed a pigment change me marked it and measured how far the pigment migrated until the next strand of pigment.

We resulted with a paper that had different colors at different distances from the base line.  If the pigments were farther from the line, then their color was lighter.  Each color represented a different pigment.  These pigments were Carotene, Xanthophyll, and Chlorophyll.   Our paper showed different pigments because of the bonding taking place between them and the paper.  Carotene was the farthest from the starting line because it is the most soluble and does not bond with the paper thus spreading along the paper the most.  Xanthophyll was next because it's bonds with the paper.  As a result, the distance was less than carotene since it had more resistance.  Chlorophyll bonds tightly to the paper resulting in even less distance from the starting point.  It also depended on their solubility.  If they were more soluble, they would travel up the paper faster.   Therefore, if another solvent was used, the Rf value would be different because of its solubility.  Finally, the reaction center would contain chlorophyll a.  All of the other pigments trap the light energy and send it to the reaction center.  

Distances from the base line.

1:Carotene, 2:Xanthophyll, 3:Chlorophyll a, 4:Chlorophyll b

Pigments climbing up the chromatography paper.  

4B: Photosynthesis, the Light Reactions

In this experiment we were trying to see if photosynthesis needs light and chloroplasts in order to occur. The chloroplasts were taken from spinach leaves and mixed the DPIP solution and placed in front of a light. Photosynthesis will become apparent when the color in the liquid begins to disappear due to when the light hits chloroplasts and boost high energy levels which then reduce DPIP.

In this experiment we were test if light and chloroplasts are both needed for photosynthesis to occur.

First we received two beakers with boiled chloroplasts and un-boiled chloroplasts. We set the spectrophotometer to 0% transmittance. Cuvette 2 was covered so no light could enter because it was the control group. Then we added three drops of un-boiled chloroplasts, 1mL of phosphate buffer and 4 mL of distilled H2O to cuvette 1.  Then to the remaining cuvettes 2, 3 and 4 we added 3 mL of distilled H2O and 1mL of DPIP. Then finally to cuvette 5 we added 3 mL and 3 drops of distilled water and 1 mL of DPIP Each cuvette was then placed in front of the light for 5, 10 then 15 minutes. Then we inserted cuvette 1 into the sample holder and set transmittance to 100%. We then measured how much light was transmitted through each of the other tubes. After that we put 3 drop of un-boiled chloroplasts into cuvette 2 and covered it with foil, but then removed the foil and put it in the spectrophotometer and measured the % of transmittance. We repeated this step for cuvette three and measured the % of transmittance as well. Then for cuvette 4 and 5 we added the un-boiled chloroplasts and measured the transmittance. Finally we compared the different % transmittance difference between boiled and un-boiled chloroplasts.

In this experiment we used DPIP to act as an electron acceptor which replaced NADP molecules. Our data shows that overall the dark cuvette had less activity than the others suggesting that the darkness resulted made it difficult to absorb the light. There was clearly an error with the no chloroplast cuvette because ideally, there would have been 100% transmittance or close to that for each trial because there were no chloroplasts to absorb the light. As the data shows, the unboiled chloroplasts/light and boiled chloroplasts/light had the most transmittance after 15 minutes which would suggest that they stopped absorbing light.  We could have run into errors when our logger pro machine froze midway through the experiment. It might not have given us a completely accurate reading. Also, the amount of time it took us to take our readings. Some cuvettes might have been exposed to the light for more time which would have an effect on the transmittance of light.

Percent Transmittance of Light through the Cuvettes

Cuvettes being exposed to light 

Cellular Respiration

Purpose: In this experiment was to measure the rate of cellular respiration in germinated and non germinated peas.

Introduction: In the experiment we were testing the rate of cellular respiration in germinated and ungerminated peas. Cellular respiration is when chemical energy is released and changed into ATP, which creates energy.

Procedure: To begin the experiment we had to measure the room temperature using a thermometer and then record the temperature in table 1.  Then we gathered 23 germinated peas and placed them into the respiration chamber. Once the CO2 shaft was in place we let the peas sit for a minute before we began to collect the data, which lasted for 10 minutes. Once a lll the data was collected a graph of CO2 gas vs. time was displayed. After that we soaked the germinated peas in ice water and then repeated the same process and graphed the data.

Discussion/Conclusion: In our experiment, our graph showed an inverse relationship between the amount of carbon dioxide and oxygen in our container.  Oxygen went down as time increased while the amount of carbon dioxide increased as time increased.  This showcases cell respiration.  The germinating seeds used the oxygen to oxidize the sugars inside and create more ATP.  They did this in order to continue germinating and allow themselves to grow.  The only way to do this is with energy.  As a result, as the seeds continued to make more and more energy, the amount of oxygen in the container decreased.  Therefore, the amount of carbon dioxide increased considering the carbons of the sugar molecules were released as carbon dioxide.  

When we submerged our seeds in water and later put them in our container, we got the same inverse relationship.  However, the amount of oxygen depleted and carbon dioxide gained did not occur at as rapid of a speed.  This is because in cellular respiration, the oxygen used is reduced to water.  Therefore, the seeds were already covered in water, making it harder for them to receive the oxygen they needed to create more energy.  This left more oxygen in the container and did not allow carbon dioxide to be created as quickly as when they were dry. 

The experiment's results could have been tampered by us not closing the container fully.  Thus, it was an open system and changes in temperature could have affected it as well as more oxygen rushing in, making our graph constant, with the amount of oxygen in the container staying the same.  When a temperature is warmer, cellular respiration occurs at a faster rate.   

Graph showing amount of oxygen and carbon dioxide in the container with dry germanating seeds.

Graph showing amount of oxygen and carbon dioxide in container filled with wet germanating seeds. 

Germanating seeds soaking in a beaker of water.

Enzyme Catalysis

Purpose:

In the experiment the purpose was to determine the rate at which a 1.5% H2O2 solution decomposes when catalyzed by the purified catalase extract.

Intro:

In the experiment enzymes play a huge role because they are also known as catalysts. A catalyst affects the rate of a chemical reaction. An enzyme-catalyst reaction occurs when the substrate attaches to the active site of the enzyme, which then causes the reaction. The more time that the enzyme has to catalyze the substrate the less of the 1.5% H2O2 solution will be used.

2B : To begin the experiment we added 10 mL of 1.5% H2O2 into a cup. Then we added 1 mL of H2O, 10 mL of H2SO4 and proceeded to mix it. Then we removed a  5 mL sample and put it into a different cup. After that we used a burette to add one drop at a time of KMnO4 to the solution. After each drop we swirled the solution until the pink or brown color remained.  We ended up getting a baseline of four after putting potassium permanganate into our solution.  Originally, we had four mL of hydrogen peroxide but it took four mL of potassium permanganate to keep the substance at a pink shade making us have a total of 8mL now.  We subtracted the amount we have now to what we started with to get our baseline of four. We put the sulfur in our solution in order to stop the enzymes from catalyzing anymore.  The pH of sulfur is too low for an enzyme to function in and ends up resulting in a denatured enzyme.  Our baseline could have been less considering we were not careful with the amount of potassium permanganate we put into our solution. This could have resulted in an excess amount of potassium permanganate.   

Solution after sulfur was added

2C:

After leaving our solution overnight, we found that 3.5 mL of hydrogen peroxide had been catalyzed.  This means that a total of 12.5% of the solution was catalyzed overnight.  This could have been altered by our setting of the room however.  The temperature of the room could have made it harder for the hydrogen peroxide to catalyze as it may not have been in its optimal temperature.   

2D:

After performing this experiment, our results ended up all over the place.  The amount of hydrogen peroxide was catalyzed using varied  time intervals.  At times, we received a positive result and at others a negative.  However, there was no pattern to our data.  This could be because of the way we handled the potassium permanganate in an uncontrolled manner.  Instead of carefully adding drop after drop, we let a stream flow into our beaker perhaps resulting in an excess of potassium permanganate.  We should have noticed that as the time interval increased, the amount of hydrogen peroxide used increased as well.  This is because the enzyme should have catalyzed the hydrogen peroxide into water and oxygen gas.  As the time increased, the enzyme had more time to do this.   

Hydrogen peroxide before potassium permanganate was added.