LNA_pglo

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University of Illinois, Urbana Champaign *

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Dec 6, 2023

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Lab 6: pGLO Transformation Lab Report Template MCB 151 LNA: pGLO Transformation Purpose The purpose of the lab is to transform and use calcium chloride to make cell membrane permeable, understanding how genes encoded with pGlo plasmid antibiotics resistance and glowing cells. At the end of this lab, students will understand the purpose of selection media, specifically ampicillin and arabinose and calculating transformation efficiency. Relevance – see rubric on Information page 6 in lab manual. Citation: Porter, A. W., Wolfson, S. J., Häggblom, M., & Young, L. Y. (2020). Microbial transformation of widely used pharmaceutical and personal care product compounds. F1000Research , 9 , F1000 Faculty Rev-130. https://doi.org/10.12688/f1000research.21827.1 Summary: The research paper discusses the microbial transformation of pharmaceutical and personal care product compounds (PPCPs) commonly found in urban-impacted environments, particularly in treated wastewater. These PPCPs can have toxic effects on wildlife exposed to contaminated water. The review focuses on the fact that Bacterial transformation plays a crucial role in the degradation of pharmaceutical and personal care product compounds (PPCPs) in various environments, especially in anoxic or low-oxygen conditions. Bacterial communities are highly diverse, and different bacterial species possess unique enzymatic capabilities. Some bacteria have evolved the ability to metabolize and transform PPCPs, converting them into less harmful compounds. This microbial diversity is essential for breaking down a wide range of PPCP molecules. Pharmaceutical and personal care products are widespread in surface waters, sediment, and soil due to various sources, including wastewater treatment plants and improper disposal. These contaminants often remain in treated effluent and can enter receiving waters or soils when biosolids are used as fertilizer, posing potential risks to the environment. Wildlife exposed to treated effluent may suffer adverse effects, such as toxicity, endocrine disruption, altered development, and changes in behavior. Bioaccumulation in aquatic organisms, particularly those intended for human consumption, is a significant concern. These issues not only impact public health but also raise concerns about ecosystem health and water quality. In summary, bacterial transformation is a promising approach for mitigating the environmental impact of PPCPs. Understanding the diversity of microbial communities and their enzymatic pathways can help researchers and environmental engineers develop effective strategies for the removal and degradation of these ubiquitous contaminants in our water systems.

Lab 6: pGLO Transformation Lab Report Template MCB 151 Procedure E. coli transformed with pGLO plasmid CalCl2. 1. Need LB agar starter plate and streak for colonies of E. coli and two tubes of . 05M of CaCl2. One tube + and – should be kept one ice. 2. Flame loop and allow to cool. 3. Lightly touch loop on empty space on colony, and gently scrap colony. 4. Transport loop to + tube and swirl to disperse bacteria. 5. Close tube and place on ice 6. Repeat process to add to negative tube. 7. Obtain 10 microliter of pGLO plasmid with pipette, be sure to look into the sample visually inspect tip. 8. Pipette into the +tube and do not add plasmid to -tube. 9. Incubate tubes and make sure fully emerged in the ice for 10 minutes. 10. Label plates with appropriate conditions 11. Transfer = and – tube to a 42 degrees Celsius water bath for 50 seconds 12. Then place back on ice for 2 minutes. 13. Remove tubes from ice and use pipette to transfer 250 microliters of LB broth to +tube using aseptic technique. 14. Repeat process to add to – tube and incubate for 10 minutes on ice. 15. Transfer 100 microliter of transformation mixture to the appropriate label LB plate. 16. Flame loop and let cool and spread bacteria over entire plate and rotate the plate, flame loop. 17. This process was to be repeated for each plate and plates were turned with gel facing up. 18. The places are stacked and placed in an incubator for 37 degrees Celsius for 16 hours.

Lab 6: pGLO Transformation Lab Report Template MCB 151 Data Presentation/Results Provide the image of your results. +pGLO/LB / amp +pGLO/LB/amp/ar a pGLO/LB/am p pGLO/LB Growth/glow ? Yes-growth No-glow Yes-growth Yes-glow No-growth No-glow Yes- growt h No- glow Why? - Cells with pGLO have antibiotic resistance therefore it grew -No glow because no arabinose - Cells with plasmid have antibiotics resistance therefore it grew - Glows because the arabinose turns on the GFP gene in the operon system - Cells with no plasmid have no antibiotics resistance therefore it cannot grow - Did not glow because no pGLO plasmid -No antibiotic and no pGLO plasmid

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Lab 6: pGLO Transformation Lab Report Template MCB 151 Conclusions 1. If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic ampicillin, then what can be inferred about the other genes on the plasmid that were involved in your transformation procedure? a. It can be inferred that the bacteria can live on the LBM plate must have the pGLO plasmid. The plasmid must express gene for ampicillin resistance. The ampicillin resistance is the protein product of the bla gene. This bla gene codes for beta-lactamase which is the protein that breaks down ampicillin. 2. From the results that you obtained; how could you prove that these changes that occurred were due to the procedure that you performed? a. To prove that these changes occurred due to the procedure performed, a comparison between the control and the experimental plates should be made. Cells treated with plasmid cannot grow on ampicillin plates where those treated with plasmid will grow on ampicillin plate. Plasmid must have gene to express ampicillin resistance. 3. Describe the evidence that indicates whether your attempt at performing a genetic transformation was successful or not successful. a. A successful experiment is seen through presence of colonies on the +pGLo LB/amp and +pGLo LB/amp/ara plates and the absence of colonies on the -pGLo LB/amp lamp. Also, for a successful experiment, the colonies on the LB/amp/ara plate should be fluoresce green. For an unsuccessful experiment, there will be an absence of colonies on the +pGLo LB/amp and +pGLo LB/amp/ara plates. This could be a result of not adding a loopful of plasmid to the +pGLo tube or not adding a colony of bacteria to the +pGLo tube. 4. From your results, can you tell if these bacteria are ampicillin resistant by looking at them on the LB plate? Explain your answer. a. You cannot tell if the bacteria are ampicillin resistant by looking at them. Both type of bacteria, those that are ampicillin resistant and those that are ampicillin sensitive, look similar when cultured. This is similar to the colonies on the LB stater plate and the colonies on the +pGLo LB/ amp plate. 5. Colony Count Determining the total number of green, fluorescent cells, using the LB/amp/ara plate . Each colony on the plate can be assumed to be derived from a single cell. As individual cells reproduce, more and more cells are formed and develop into what is termed a colony. The most direct way to determine the total number of green, fluorescent cells is to count the colonies on the plate.

Lab 6: pGLO Transformation Lab Report Template MCB 151 Enter that number here ⇒ 138 6. Transformation Efficiency Calculation Determining the amount of pGLO plasmid DNA in the bacterial cells spread on the LB/amp/ara plate. We need two pieces of information to find out the amount of DNA (pGLO) in the bacterial cells spread on the LB/amp/ara plate in this experiment. (i) What was the total amount of DNA we began the experiment with, a. The total amount of DNA we began the experiment with was 0.8 micrograms. (ii) What fraction of DNA (in the bacteria) were spread onto the LB/amp/ara plates? a. The fraction of DNA in the bacteria that actually got spread onto the LB/amp/ara plate was 0.16 micrograms of pGLo. After you calculate this data, you will need to multiply the total amount of pGLO plasmid DNA used in this experiment by the fraction of DNA you spread on the LB/amp/ara plate. The answer to this multiplication will tell you the amount of pGLO plasmid DNA in the bacterial cells that were spread on the LB/amp/ara plate. a. Determining the total amount of DNA The total amount of pGLO plasmid DNA we began with is equal to the product of the concentration and the total volume used, or DNA (μg) = (concentration of DNA (μg/μl) x (volume of DNA in μl) In this experiment, you used 10 μl of pGLO at a concentration of 0.08 μg/μl. This means that each microliter of solution contained 0.08 μg of pGLO DNA. Calculate the total amount of DNA used in this experiment. Enter that number here ⇒ 0.08 μg/μl * 10 μl = .8 μg How will you use this piece of information? This number will be multiplied by the fraction of DNA used to determine the total amount of DNA spread on the agar plate. Determining the fraction of pGLO plasmid DNA (in the bacteria) that were spread onto the LB/amp/ara plate. Since not all the pGLO plasmid DNA you added to the bacterial cells will be transferred to the agar plate, you need to find out what fraction of the DNA was spread onto the LB/amp/ara plate. To do this, divide the volume of DNA you spread on the LB/amp/ara plate by the total volume of liquid in the test tube containing the DNA. A formula for this statement is: Fraction of DNA used = Volume spread on LB/amp plate Total volume in test tube

Lab 6: pGLO Transformation Lab Report Template MCB 151 You spread 100 μl of cells containing pGLO DNA from a test tube containing a total volume of 510 μl of solution. Do you remember why there is 510 μl total solution? Look in the laboratory procedure and locate all the steps where you added liquid to the reaction tube. Add the volumes. Use the above formula to calculate the fraction of DNA you spread on the LB/amp/ara plate. Enter that number here ⇒ 500 μl or total volume + 10 μl = 510 μl 100 μl /510 μl = .2 μl How will you use this piece of information? This number will be multiplied by the amount of DNA used to calculate the amount of DNA spread on an agar plate. So, how many micrograms of DNA did you spread on the LB/amp/ara plates? To answer this question, you will need to multiply the total amount of DNA used in this experiment by the fraction of DNA you spread on the LB/amp/ara plate. pGLO DNA spread (μg) = Total amount of DNA used (μg) x fraction of DNA Enter that number here ⇒ 0.8 μg * 0.2 μl = 0.16 μg What will this number tell you? This number tells you how much DNA was spread on the agar plate. Look at all your calculations above. Decide which of the numbers you calculated belong in the table below. Fill in the following table: Number of colonies on LB/amp/ara plate 138 Micrograms of pGLO DNA spread on the plates 0.16 μg Now use the data in the table to calculate the efficiency of the pGLO transformation. Transformation efficiency = Total number of cells growing on the agar plate Amount of DNA spread on the agar plate. Enter that number here ⇒ 862.5 colonies/ μg

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