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Bacterial Transformation: Manipulation of E. Coli.

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by

Jin Chen

on 9 December 2013

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Transcript of Bacterial Transformation: Manipulation of E. Coli.

Discussion
Looking back at our results, I think the experiment was somewhat of a success. We managed to get bacteria to glow and the ones that weren't supposed to didn't. We found that the dish that did not contain ampicillin had the most growth. According to the procedures, this is correct because ampicillin is an antibiotic which kills bacteria. Therefore it would make sense to have most bacteria growth in a dish without ampicillin. To further support this claim, the bacteria in the -pGLO/LB/AMP did not have any bacterial growth meaning ampicillin must have restricted cell growth. In the petri dish with +pGLO/LB/AMP which took in the pGLO gene shown growth but not a large amount meaning the bacteria that didn't take in the gene were killed. In the petri dish +pGLO/LB/AMP/ARA, the bacteria glowed, meaning it took in the pGLO gene and with the addition of arabinose, it was able to glow AND reproduce in a medium with ampicillin. Overall, I think this lab was a success because +pGLO/LB/AMP and +pGLO/LB/AMP/ARA were able to grow in a medium with ampicillin and that +pGLO/LB/AMP/ARA's bacteria colonies were able to glow under ultraviolet light. Judging from the results, I can say we successfully manipulated E. coli's genetic information, allowing it to glow and become resistant to antibiotics and because our results correspond with our hypothesis, we will accept our hypothesis.
Conclusion
The purpose of this lab was to find out if we can successfully manipulate E. coli but altering it's DNA to make it glow and make it resistant to ampicillin through the process of bacterial transformation. This in relation to the field of science is significant because it teaches us about how we can genetically modify organisms by the use of plasmids, restriction enzymes and DNA ligase. Scientists can insert desired traits into plasmids and then placed into bacteria to inherit the trait. This opens many doors in the field of medicine and agriculture. By being able to manipulate genetic information, scientists can create new medicines and genetically alter crops so that they withstand the cold or insects and more. Things we could change if we were to do this experiment again to make it more accurate is to do it in a more sterile environment to avoid contamination and not talk when plating the bacteria.
Bibliography
Background
Deoxyribose Nucleic Acid (DNA) is the molecular genetic code that hold instructions used in development and functions in all known living things (Wikipedia). Genetic information can also change due to mutations or through biotechnology. When the DNA of a cell changes, the RNA proteins they produce would change along with it. Which in turn changes how cells function. (Investigation 8 - Biotechnology: Bacterial Transformation). However, it wasn't easy getting to where we are today with our knowledge of DNA. It took many years and many experiments to find how DNA worked. T.H Morgan suggested that genes are located on chromosomes. His works included fruit flies. Frederick Griffith experimented with mice and pneumonia in which he concluded that there was a transforming factor. Avery, McCarty and MacCleod followed Griffith's footsteps and concluded that DNA is the substance that causes bacterial transformation. All of the experiments are then confirmed by Hershey and Chase with their famous "Blender Experiment". Biotechnology was not possible until the discovery of Restriction Enzymes by Werner Arber, Daniel Nathans and Hamilton O. Smith. Using restriction enzymes, scientists can cut up DNA and glue it together with DNA of a desired trait. This is done using plasmids. When the desired trait is integrated into the plasmid, the bacterium will then absorb the plasmid and incorporate it's DNA to itself. However, the discovery of biotechnology raised many questionable issues such as safety of it's use.
Results/Observations
The petri dish with -pGLO/LB had the most bacterial growth. It was impossible to count but they didn't glow under UV lighting. In the -pGLO/LB/AMP dish, there was no bacterial growth meaning the ampicillin prevented it from growing. In the +pGLO/LB/AMP dish, there was bacterial growth and was able to count. We counted approximately 250 colonies. However, none of the bacterial glow but it was resistant to the ampicillin. And our last +pGLO/LB/AMP/ARA dish, we were able to see it glow under UV light meaning the experiment was a success and we counted approximately 150 colonies. However, we thought that there would be more colonies +pGLO samples. This could've been due to contamination. This resulted in a low transformational efficiency of 250 where it should've been 800.
Abstract
Bacterial transformation is the process of altering a cell's genome by incorporating it with a desired trait from another source. This is done by first altering plasmids, which the bacterium will take up later on. Our primary purpose of this experiment is to find out if we can successfully manipulate the bacteria E. coli to express a glowing gene and to become resistant to the antibiotic Ampicillin. For this experiment, we will be following the procedures provided by the Student Manual - pGLO Transformation. First of all, we were to prepare two test tubes consisting of "+" or "-" solutions. We then added bacteria to both solutions and pGLO plasmid DNA into the "+" solution only. We then added the prepared bacteria to multiple dishes. They are then placed into the incubator to allow it to grow and multiply. After the lab process was done, our results showed that with the addition of arabinose sugar, the bacteria was able to glow under ultraviolet light. However, one out the four dishes didn't have bacteria on it. This is because the bacteria that was on the dish was not resistant to ampicillin. It wasn't resistant to ampicillin because it was not given the pGLO plasmid DNA. The pGLO plasmid encodes for the fluorescent and resistance to ampilcillin gene. This experiment is important because it proves that we can manipulate a bacteria's DNA and by being able to manipulate it's DNA to give new functions, we can use this technology to benefit ourselves in areas like medicine and crops.
Bacterial Transformation Lab:
Manipulation of E. Coli.

Introduction
The design of this lab is to try and transform the bacteria E. coli to express new traits by altering it's genetic code. In this experiment, we will be trying to make the bacteria, E. coli glow under ultraviolet light and have it survive in a petri dish with ampicillin. Our null hypothesis for this experiment would be that once we altered the DNA genome, there will be no change in the bacteria's phenotype nor that it will grow in a petri dish. Our alternative hypothesis is that the pGLO gene will get integrated into the bacteria successfully and will glow under ultraviolet light and able to grow and multiply in a petri dish with ampicillin. Our predictions for this experiment is that -pGLO/LB will have the most bacterial growth because it does not contain ampicillin in the petri dish it's in. -pGLO/LB/AMP will have no bacterial growth because the ampicillin will just kill the bacteria. +pGLO/LB/AMP will have some growth due to the addition of the pGLO plasmid DNA. Some will grow because not all the bacteria will incorporate the resistance to ampicillin. Last but not least, +pGLO/LB/AMP/ARA will be the bacteria that glows under ultraviolet light because of the arabinose sugar that triggers the fluorescent gene to be expressed. The purpose of this experiment is to see if we can manipulate E. coli to glow and become resistant to ampicillin by inserting a gene into it that it doesn't normally have.
Methods
Refer to the materials and procedure found in the A
P Biology
Investigative Labs Manual (College Board, 2012),
Investigation 8 -
Biotechnology: Bacterial Transformation
"Investigation 8- Biotechnology: Bacterial Transformation Lab Procedures

"AP Biology Labs - Part 2." YouTube. YouTube, 10 May 2013. Web. 08 Dec. 2013.


"Bacterial Transformation." Bacterial Transformation. N.p., n.d. Web. 07 Dec. 2013.
-pGLO/LB
Numerous bacterial colonies to count. (Bacteria did not glow)
-pGLO/LB/AMP
No colonies
+pGLO/LB/AMP
Approximately 250 colonies. (Bacteria did not glow)
+pGLO/LB/AMP/ARA
Approximately 150 colonies (bacteria glowed). Colonies crowded around the edges.
Results to literature/theoretical behaviour
With wonderful experiments from the past, drug companies can use this knowledge to further produce more helpful drugs to help society. For example, they can manipulate bacteria to make insulin where it is then harvested and purified into medicine.
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