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Nicholas Mack 3 February 2016

pGLO TRANSFORMATION LAB REPORT

Introduction: The purpose of this lab was to observe the effects of the pGLO plasmid on various colonies of E. coli bacteria. pGLO is a genetically modified plasmid that primarily contains three genes (with the origin of replication). The first is an ampicillin (antibiotic) resistance gene that allows the genetically modified bacteria to grow in this antibiotic. The second is the GFP gene that codes for the green fluorescent protein, which, when in the right conditions, causes bacteria to emulate green light. The third gene, araC, acts as a “trigger” to activate the GFP gene when the bacteria are in an environment with this type of sugar. In this experiment, students transferred the modified plasmid to the E. coli via transformation: the process by which a plasmid is removed from its host and placed into a foreign bacteria cell. Once the plasmid is inside the new cell, the bacteria are able to recognize the genes encoded in the plasmid and express a desired phenotype. For this lab specifically, student transferred the modified plasmid to the E. coli by using a transformation solution containing CaCl. A process called heat shock was then used to insert the plasmid into the E. Coli. Four different growth plates were used for bacteria growth to simulate different groswing conditions. The first plate (pGLO / LB) did not have ampicillin and contained bacteria that did not have the plasmid. There should be a lot of growth on this plate because the bacteria will be able to feed off of the broth (LB). However, the colonies should not glow because, in this case, a repressor protein will be bound next to the promoter (starting point) of the GFP gene as an acting operon, causing transcription of that gene to stop until ARA is present. This is an example of how the GFP gene is regulated. The second plate (-pGLO / LB / AMP) should not exhibit any growth because, since the bacteria do not have the pGLO plasmid, they are not resistant to ampicillin, causing no growth. The third plate (+pGLO/ LB / AMP) should exhibit moderate growth because the bacteria do contain the plasmid, allowing them to grow on ampicillin and feed off of LB. However, the color of these cells should remain white because the repressor will still be bounded to the operator due to the absence of the sugar, causing transcription of the GDP gene from its promoter to be

turned off. The fourth plate (+pGLO / LB / AMP / ARA) should exhibit bacteria that are glowing because, with ARA present, an ARA inducer molecule will be able to bind to the repressor to inactivate it, allowing transcription of the GFP to commence. With this experiment, students will have a better understanding of how plasmids and gene regulation work. Results: Expected:

+ Source: https://sites.google.com/a/pvlearners.net/aishwarya-karlapudi/blog-1

**Note: DNA + and DNA – are equivalent to saying pGLO + and pGLO -.

Actual: Observations of E.Coli Plates With and Without pGLO Plate Name - pGLO / LB

Observations 1. There is a lot of growth. A lawn has formed. Bacteria colonies are clustered.

-pGLO / LB / AMP

2. Color is white with and w / out UV. 1. As expected, there is no growth at all. 2. None with and w / out UV light

+ pGLO / LB / AMP

(obviously). 1. Moderate growth, though the colonies are not as large as – pGLO / LB. More scattered throughout plate. 2. Color is white with and w /out UV 1. There is a lot of growth. Bacteria

+ pGLO / LB / AMP / ARA

colonies are clustered. 2. Color of the bacteria appears to be white without UV light and green and glowing with UV light *Pictures of All Plates On Separate Document Conclusion: After concluding this experiment, the group knew that the transformation was successful because the E. coli glowed in the plate with + pGLO / LB / AMP / ARA, indicating that the bacteria successfully received the plasmid with the GFP and AMP genes. Because the environment contained the arabinose sugar, an ARA inducer molecule was able to inactivate the repressor and allow transcription of the inserted GTP molecule to commence. Other evidence includes the fact that the bacteria in the + pGLO / LB /

AMP plate successfully grew because they had the antibiotic resistance gene. The -pGLO / LB / AMP plate indicates that without this antibiotic resistance gene, the bacteria would not be able to survive in an environment with ampicillin. In terms of improvement, what the group could do better next time is to incubate the test tubes containing LB nutrient broth for the specified amount of time. During the experiment, the group did not incubate these tubes for enough time, which could have affected their results. The group will be sure to improve their timing for the next experiment. In this experiment, the group successfully produced genetically modified bacteria that exhibited desired phenotypes. Each E. coli cell with the inserted plasmid was able to regulate the expression of the GDP by utilizing an inducer molecule. Based on the results from this experiment, there is an incredible advantage to turning on and off genes. If a certain chemical or substance is present in an organism’s environment that the organism’s cells need to digest, then a particular gene coding for digestion can be turned by inactivating its repressor. When these enzymes are not needed, the repressor can be simply activated, turning off transcription. Thus, the overall advantage of turning on and off genes is that it conserves energy. By conserving energy, a cell can prioritize its duties and become more efficient. There are numerous applications of transforming bacteria. One potential application is that transforming bacteria can be used in bioremediation to eliminate pollution in an environment, such as during an oil spill. Using genetic engineering, a plasmid that contains genes for pollutant digestion can be inserted into a specific type of bacteria. The genetically modified bacteria can be then introduced to a specific environment where it will be able to eliminate toxins and pollutants. In the scenario of an oil spill, genetically modified bacteria can be used to break down “hydrocarbons found in oil.”

Source: http://katiebobatey0.tripod.com/bacterialtransformation/id1.html