pGLO Transformation Lab

Hello and welcome to our fabulous P.Glo lab done by Aliya, Emma, Cara, and Ally (your fav senior ladies who value and appreciate you Mr.Filipek) 

Purpose

The purpose of this experiment was to perform and prove genetic transformation which is insertion of a gene into an organism in order to change it's trait. In this experiment we were testing the insertion of the GFP(green fluorescent protein) gene into the P.Glo plasmid of the E.coli cells. 

Introduction

In this experiment, E.coli is a bacteria that is commonly found inside of mammals and birds and is used to breakdown food into small molecules that can be transferred into your blood. It is a very basic prokaryotic cell and only lives a short time. Most types of E.coli are good for you but there are a very few select types that can be poisonous to humans such as E.coli found in raw meat. Genetic transformation, in this experiment is used to transfer new DNA from the GFP to plasmid of the E.coli cells. A plasmid is a small circular piece of DNA where the GFP can be inserted in order to show new traits within the cell. The insertion of GFP into the pGlo makes the cell resistant to antibiotics.

Methods

We started of by using two micro test tubes one of +pGlo (containing pGlo) and one of -pGlo (not containing pGlo). We then placed 250 microliters of CaCl2 into each tube. Next the two tubes we're placed on ice. After this, we put a sample of the E.coli bacteria into each tube using a loop. Immediately, we inserted pGlo plasmid DNA into the positive pGlo tube but not in the negative pGlo tube. Then we incubated (let them sit on ice) for 10 minutes. Right after this, we performed a heat shock on both tubes. Next, we put 250 microliters of LB nutrient broth in both tubes. We then placed 100 microliters of the solution from each tubes onto 2 Agar plates each, resultin in 4 total plates. One plate of +PGlo LB/amp, one of +pGlo LB/amp/ara, one of -pGlo LB/amp, and one of -pGlo LB.

Data

Graphs and charts

Drawn-out representations of our Agar plates and their contents after the lab was completed

Discussion

Our results from this lab were a direct result of the proccess of genetic transformation. Some of the dishes expressed the pGLO trait while others did not because of this proccess. Those that did express the trait those that expressed the "glow in the dark" trait were those with pGLO and LB/amp. The plate with LB/amp/ara contained more  colonies because of the sugar that was added to the plate that helped with growth. If we were to make adjustments to this lab, we would put more of each substance of each plate in order to see more colonies then what we did the first time. It's difficult to tell whether our results are completely accurate due to how little of data that we have. As predicted, the petri dishes that had the GFP inserted into the plasmid glowed in the dark, while the petri dishes without the plasmid did not. The insertion of the GFP into the plasmid then makes it resistant to antibiotic. The LB plate has no growth because it has no protein to resist antibiotic. 

Conclusion 

Through this lab we were able to prove that pGLO causes the bacteria to be able to resist the antibiotic. 

References 

http://scienceforkids.kidipede.com/biology/cells/ecoli.htm

Pre-lab selfie (super excited)

Receiving the translation solution (super fun)

Icing the solutions (super chill) 

Getting the E.coli (super thrilling)

E.COLI (yay)

Heat killing the solutions (super amazing)

Aliya and Emma in the action (feat. Tape and coffee)

Petri dish! (Super scientific)

Mixing! (Super cool and fun)

All the Petri dishes in their final stage of the lab! ( super exciting and biological) 

+pGlo LB/amp/ara

-pGlo LB

+pGlo LB/amp

-pGlo LB/amp

How To: Extract DNA From a Strawberry

Purpose: Hello! This was one of our favorite labs because we all love strawberries a whole lot. The purpose of this experiment was to see if there was a way to extract a strand of DNA from one whole fresh strawberry. 

Introduction: DNA is found in all living organisms and in this lab our goal was to extract a strand/fibers of DNA from the strawberry. 

Methods: First we mashed up the strawberry in a ziplock bag in order to make the DNA more extractable. Then we mixed the mashed up strawberries with the extraction buffer (the soapy water mix). After letting the mixture soak through a coffee filter into a graduated cylinder, we collected the liquid after a few minutes. We then added a few drops of ice cold alcohol to the strawberry and soapy water solution. We made sure to let the alcohol drip down the side of the graduated cylinder instead of dropping it straight in. After a minute or two a white cloudy layer appeared. This was the DNA! After stirring the pipet in the mixture, the DNA climbers could be pulled out and examined.  

Discussion: Our results from this lab successfully proved this lab true. DNA could be successfully extracted from a strawberry as long as we followed the correct steps, which we eventually did. The only thing that went wrong was our first attempt at the lab. We first mixed the mashed up strawberry with the ice cold alcohol to find nothing so we knew then that we had to approach the lab differently. Eventually we figured it out and successfully extracted the DNA from the strawberry. If we were to improve our tactics and techniques in this experiment, we would have not first mixed the alcohol with the strawberry solution. The results of our experiment proved that DNA fibers/strands are found in strawberries. 

Conclusion: To conclude this experiment, DNA can be extracted from raw strawberries as long as it is mixed with an extraction buffer, filtered through a coffee filter and mixed with alcohol. Our results from our lab support this conclusion. 

References: 

DNA Extraction From Straberries (Schoology) 

Pictures

The mashed up strawberries!!!

Adding the alcohol to the solution!!!

Extracting the DNA (feat. Mr. Filipek) !!!

Cell Communication Lab

Purpose

The purpose of this experiment was to observe cell communication between two strains of yeast (A-type and Alpha-type), both of the two being opposite sexes.

Introduction

Cell Communication is essential for organisms in order for signals to turn into responses within the cell. In this lab we will be studying methods by which yeast cells partake in cell communication. It is our job to determine what type of cell signaling  these yeast cells use and how these cells send signals to each other. We will complete this lab with the use of yeast, Petri dishes, and microscopes. 

Methods

We began this lab by separating samples of the a-type, the alpha-type, and a mixture of the two in order to determine the reproduction rate and how the yeasts go about it asexually as well as sexually. We placed each of the wet yeasts in its own Petri dishes and let them sit over night. Before we wrapped up the lab on the first day we measured the yeasts under a microscope. We put broth in the test tubes and placed them into an incubator overnight,. The next day we scraped the dried yeast off the petri dishes and mixed if with water and put each yeast on a glass slide and put it under a microscope. We then measured our data to find the change in yeast cells over time. 

Mixed Yeast (Day 1)

A Type Yeast (Day 1)

Alpha Type Yeast (Day 1)

Alpha-Type Dry (Day 2)

Mixed Dry (Day 2)

A-type (Day 2)

Mixed Wet (Day 2)

Data

Mixed Yeast Culture Data

Alpha-Type Yeast Culture Data

A-Type Yeast Culture Data Table

Graphs and Charts

Graph of the percent change of yeast cells over time (haploid and diploid)

Discussion

In the Cell Communication Lab we measured the difference between the different yeasts (alpha type, A-type, and mixed) to see their sexual/asexual production. We observed that there was not much difference between the a-type and the alpha-type strains of yeast because they are both able to reproduce asexually, there was not much difference in budding of the cells. But, when the alpha-type and A-type were mixed together it is observed that right away there is already diploid zygotes and diploids zygote budding from the cells beginning to grow towards each other because they are opposite sexes. This goes to prove that though yeasts are able to reproduce asexually, bringing the two opposite sexes together causes cellular communication to occur by the sending and receiving of signals to reproduce at a more rapid rate. One thing that could be done differently in this experiment was counting of the different haploid and budding haploid cells. More test trials would have been more efficient but we spent a majority of time trying to get pictures to accurately represent our data. Another thing would be to use different settings on the microscope to get a closer look at the cells and be able to examine the mixture culture closer. 

Conclusion

In this experiment we see that the most favorable condition for yeast reproduction is when yeasts are mixed with a-type strains and alpha-type strains. This is because of the cell's ability to to communicate through signaling between different sexes and as long as the environment is favorable, diploid zygotes are formed and divide.

References

All I Want For Christmas Is Mitosis

Gettin' into the Holiday spirit!

Plant Pigmentation and Photosynthesis Lab

4A Plant Pigment Chromatography

Purpose

The purpose of this experiment was to use Chromatography paper to identify plant pigments in spinach cells. We were testing to see if certain types of pigments were in spinach and the solubility of these pigments (Rf values). The relationship between the pigment and the solvent is the Rf value.

Introduction

The different pigments plants contain are carotene, xanthophyll, chlorophyll a and chlorophyll b. Paper Chromatography is used to separate the molecules because of the varying solubility of molecules in a certain solvent. It does this by separating the pigments using capillary action. This happens because solvent molecules are attracted to the paper as well as each other. When the solvent moves up the paper, it carries the pigments with it.  The distance the pigments travel is dependent on their solubility.  

Methods

In this lab we began by cutting a strip of chromatography paper into a pencil shape. (A long strip with a pointed end). Next, we scraped spinach cells off of a spinach leaf with a coin and dragged them across the chromatography paper, making sure that this green line did not go past the pointed end of the chromatography paper. We then hooked the chromatography paper onto a hook on the bottom of a stopper that was to go in a 50-ml graduated cylinder. The graduated cylinder had 1 cm of solvent in the bottom of it. We then made sure that the solvent only touched the pointed end and did not emerge the strip portion. The rest of the lab we let this chromatography paper sit and waited to record our results when the period ended, after the chromatography paper had diffused all of the spinach's pigments up the paper. 

Rubbing spinach cells onto chromatography paper

Placing the paper in into the test tube that holds the solution

Waiting..

Waiting...

Still waiting....

Measuring the distances traveled!

Measuring the distance of each band number 6.3-.6

Data

Graphs and Charts

Discussion

Our results contained the Rf values for 4 specific pigments found in spinach leaves: Carotene, Xanthophyll, Chlorophyll a, and Chlorophyll b. These results were directly related to how far each of them traveled up the chromatography paper. Each pigment was able to travel different distances up the paper because each pigment had different solubility levels and the chromatography paper is able to show this. A trend found in our data was the relationship between the distance of each pigment traveled and the Rf values of each of the same pigments. As a result if this, a conclusion can be made that the further the distance a pigment travels up the chromatography paper, the higher it's solubility is. The ones who traveled the furthest had the lowest Rf values and vice versa. These results were accurate and support our answer. If we were to change anything or re-do certain aspects of this lab we would have correctly measured the pigment's distances during the actual lab. Instead we just took pictures of the distances and referred to them because we were running out of tine by the end of this lab. Other than this, our lab was successful. Our results support this conclusion.

Conclusion

To conclude this experiment, spinach contained these 4 pigments: Carotene, Xanthophyll, Chlorophyll a, and Chlorophyll b. The solubility of these pigments were a directly related to how far the pigment traveled. Chlorophyll b had the lowest Rf value and traveled the shortest distance while Xanthophyll had the highest Rf value and traveled the furthest. Our data correctly supports this answer. 

References

http://www.gcisd-k12.org/cms/lib4/TX01000829/Centricity/Domain/783/2014%20Paper%20Chromatography%20of%20a%20Spinach%20Leaf%20Lab.pptx

4B Photosynthesis/ The Light Reaction

Purpose

The purpose of this experiment was to test whether light and chloroplasts are required in order for light reaction to occur.

Introduction

Many factors can either inhibit, help, or have no effect on Photosynthesis (light reactions). This lab was conducted to study this. DPIP, a compound posing as an electron acceptor, water, a phosphate buffer, and a spectrophotometer were used in this lab to reach the answer to this question. A dye-reduction technique is used in this experiment to study photosynthesis. 

Methods

To begin in this lab, we added the correct amount of DPIP, distilled H20, and phosphate buffer to each of the 5 cuvettes. The only one that did not contain DPIP was the first one. We made sure to wrap cuvette 2 in tin foil because no light was supposed to enter this cuvette. Each of the 5 cuvettes were measured in the spectrometer right after their mixtures were created. After each of these results were recorded, a 5, 10, and 15 minute time period was measured for each cuvette again. While one cuvette was in the spectrometer, the other 4 (except for number 2) were placed in front of a heat sink filled with water that was placed in front of a light.The heat sink's purpose was to absorb the heat so the cuvettes would experience no heat. After each of the 5, 10, and 15 minutes of the cuvettes were finished, we recorded our results.  

Measuring DPIP

Filling a cuvette with DPIP

Still filling

Exposing the cuvettes to light

All of the materials used

Unboiled chloroplasts 

Data

                                    % Transmittance

Graphs and Charts

Discussion

Unboiled/dark and Unboiled/light both had a significant increase in transmittance within the first five minutes of the experiment which shows that photosynthesis occurs in both light and dark environments while the chloroplasts are still alive. Contrarily, the chloroplasts that were boiled had very little growth as time went on which showed that the denatured chloroplasts hardly undergo photosynthesis.  The change in transmittance that was apparent with the boiled chloroplasts may be due to some of the sample not being fully denatured.  If this experiment were to be done again, it would be beneficial to make sure all of the chloroplasts are fully denatured so that there would be even less photosynthesis occurring within the boiled chloroplasts.  At the same time, it may be inevitable to have a portion of the sample could survive being boiled.  This comparison also shows that the light has much less of an impact on photosynthesis occurring than the nature of the chloroplasts, as those which were simply not exposed to the light still transformed, while the boiled chloroplasts were incapable of undergoing photosynthesis. The results we obtained are in sync with what we thought would happen because in order for plants to go through photosynthesis there needs to be a presence of light (in order to start the light/dark reactions) and chloroplasts, in order to absorb the light. If we conducted the experiment again, in order to see a more significant transmittance change, we would need to be more careful with how we mix or cuvettes as well as being more persistence with how much of each substance is being tested. Also, testing each cuvette between a certain time period rather than so close together so the time we collect our data is more exact, because that affects the transmittance measured.

Conclusion

To conclude this experiment, light does not have as big of an impact on photosynthesis as chloroplasts do. Our data generally supports this theory except for the slight differences in the transmittance levels of the boiled chloroplasts. In order for our data to completely support our theory, all of these levels should have been exactly the same. 

References

http://www.nature.com/scitable/content/the-light-and-dark-reactions-in-the-14705803

Cell Respiration Lab

Purpose

The purpose of this experiment was to observe the concentrations of carbon dioxide within a chamber during cellular respiration while various factors impacted in the process.  The independent variables were the temperature changes, and the germinating/non-germinating peas.  The dependent variables were the amounts of carbon dioxide produced and the rates of cellular respiration.

Introduction

Cellular respiration is the process cells undergo to break up sugars into a form that they can utilize as energy.  Cellular respiration takes in food and uses it to create ATP, a chemical the cell uses for energy. The process of cellular respiration consists of many different cycles, including the citric acid cycle.  During the citric acid cycle, carbon dioxide is produced and then later released as waste.

Methods

In this lab we tested the amount of oxygen consumption with the use of the devices. The beans, seeds, or glass beads were placed into the clear plastic container and we put the carbon dioxide and oxygen detectors into the openings on top of the container. The measures of these two gases appeared on the screen of the device. We then transferred this data onto each of our iPads.

Data 

The data from this lab explains the relationship between oxygen consumption and cellular respiration. 

Graphs and Charts

Discussion

It's evident that cellular respiration occurred in the peas. For the non germinated peas at room temperature, there was an increase in carbon dioxide while oxygen decreased. This means cellular respiration was at work and there was an exchange of the two gases. Similarly, for the non germinated peas with ice, there was an increase in carbon dioxide while the oxygen decreased. This shows non germinated peas were effected more then germinated peas. For the germinated peas in both room temperature and ice, there was little change in the oxygen and carbon dioxide levels. Germinated peas undergo cellular respiration because it's necessary for them to obtain energy this way. 

Conclusion

The germinating peas had the greatest celluar respiration of oxygen. The germinating peas had a faster process of cellular respiration than the non-germinating peas. The non-germinating peas required less energy, so because the dry peas were non-germinateing they had slower cellular respiration. The higher temperature caused the cellular respiration to occur faster and this caused a greater consumption of oxygen. The beads had no cellular respiration because they were unable to undergo the same process. 

 References

Enzyme Catalysis Lab

Purpose

The purpose of this experiment is to observe the change of hydrogen peroxide to water and oxygen gas by an enzyme catalase as well as to measure how much oxygen was made and calculate the rate of the enzyme-catalyzed reaction.

Introduction

Enzymes are proteins made from living cells that acts as a catalyst which affects the rate of a chemical reaction. In an enzyme-catalyzed reaction, the substrate binds  the active site of the enzyme. Each enzyme is specific for a certain reaction because each into acid sequence is unique and enzyme can be affected by the salt concentration, pH, temperature and activations and inhibitors.

Methods

We first created a base line in order to determine the hydrogen peroxide that is initially in the solution and use that base line to see the uncatalzyed rate of decomposition versus the catalyzed rate of decomposition. Sulfuric acid is added to inhibit the enzymes because of the acidic environment and cause it to stop reacting. After adding sulfuric acid, a 5-mL sample is taken in order to be titrated to see the catalyzed rate or change over time. potassium permanganate is added drop by drop from a burette until the solution is turned a pink or brown color.

Cups to be mixed fit various amounts of time.

When testing the baseline sample, there is initially a brown/pink color, but with some stirring, that hue disappears (pictures 1 & 2).  Once some potassium permanganate is in the solution and the color doesn't dissolve, the reaction has reached the end (picture 3).

Adding catalase extract (yeast) to begin reaction.

Adding Sulfuric Acid to stop reaction.

Taking a 5mL sample to test with potassium permanganate.

Test with potassium permanganate until there is a consistent pink or brown color.


Data

Discussion

The overall objective of the experiment was to observe the relationship between Hydrogen Peroxide and the catalase extract.  More specifically, observing the transformation of Hydrogen Peroxide to water and oxygen gas due to an enzyme catalase (in this case, the enzyme catalase was yeast).  Each time another aspect of the experiment was conducted, aka the catalase was left in the Hydrogen Peroxide for a longer period of time, the results varied.  From ten seconds to thirty seconds to sixty seconds, the amount of Potassium Permanganate consumed was sporadic, going from 3.2mL to 2.8mL, then back up to 3mL.  While these results were all very close, no true patterns were apparent.  In general, the amount of Potassium Permanganate consumed decreased between ten seconds and 360 seconds of the Hydrogen Peroxide solutions being mixed with the yeast.  The same applies to the amount of Hydrogen Peroxide used throughout the reaction: there was a general trend of decreasing of the usage of the solution as the length of time increased--although there were multiple instances in which the data did not follow the trend exactly.  For example, from ten seconds to thirty seconds to sixty seconds, the amount of Hydrogen Peroxide used initially decreased, but then increased right after.  The interval of time from 0-10 seconds had the highest rate because that is when there were the most enzymes and substrates present.  It is clear that the interval of 180-360 seconds had the lowest rate, which is because by the time the solution had been being mixed for so long, most of the enzymes and substrates were essentially used up, causing the reaction to plateau.  Since there was a relatively sure and consistent amount of the Hydrogen Peroxide, enzyme catalase, and Sulfuric Acid being used in each sample, and each time a titration took place, it was always with 5mL of the entire solution, the inconsistencies most likely come from the titration part of the experiment.  It is very easy to let a little too much Potassium Permanganate through the titration at once.  If too much is released when the pink or brown color was already sticking around, the results could easily be impacted.  To improve this experiment in the future, it would be important to make sure all measurements are accurate--from the amount of each solution used, to the amount of time each solution is mixed for.

Conclusion

Through this lab we observe how the catalase increases rate of decomposing of hydrogen peroxide. We see how hydrogen peroxide and enzyme catalase are able to work together. With the different time trials we are able to see how the enzyme breaks down hydrogen peroxide over a period of time. Uncatalzyed decomposition is slower than catalyzed decomposition of hydrogen peroxide. The catabolic process helps to speed up decomposition and break down, an example of our liver's ability to break down toxins.