Transformation of e coli with pgal lab report. Pgal Transformation Complete Lab Revised 2022-12-07

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Transformation is a process by which genetic material is introduced into a host cell, allowing it to express new traits or functions. One common method for transformation is the use of Escherichia coli (E. coli) as a host cell and a plasmid containing the desired genetic material. In this lab report, we will describe the transformation of E. coli with a plasmid containing the gene for production of galactose (pgal).

To begin the transformation, a competent strain of E. coli was prepared by chemically treating the cells to make them more receptive to the uptake of foreign DNA. The competent cells were then mixed with the pgal plasmid, which had been purified from a bacterial culture. The mixture was incubated for a brief period of time to allow the plasmid to enter the cells.

After the incubation, the cells were plated on a nutrient agar medium containing the antibiotic ampicillin. Ampicillin resistance is a trait conferred by the pgal plasmid, so only cells that have taken up the plasmid will be able to grow on this medium. The cells were incubated at 37°C for 24 hours, allowing them to form colonies.

The next day, the colonies were counted and the transformation efficiency was calculated by dividing the number of colonies by the number of cells plated. A high transformation efficiency indicates that a large proportion of the cells were able to take up the plasmid and express the ampicillin resistance trait.

To confirm that the transformation was successful and the pgal plasmid was present in the cells, we performed a polymerase chain reaction (PCR) to amplify the gene encoding the enzyme responsible for production of galactose. The PCR product was then separated by electrophoresis and the resulting band was visualized under ultraviolet light. A band of the expected size indicated that the pgal gene was present in the transformed cells.

In conclusion, the transformation of E. coli with the pgal plasmid was successful, as indicated by the high transformation efficiency and the presence of the pgal gene in the cells as confirmed by PCR. This transformation demonstrates the versatility of E. coli as a host cell for the expression of foreign genes and the usefulness of plasmids as a means of introducing these genes into the host cells.

typemoon.org Lab Report

transformation of e coli with pgal lab report

The number of satellite colonies increases if the concentration of ampicillin is low or if the plates have incubated for extended times. Quantitative Analysis of DNA Fragment Sizes If you were on trial or were trying to identify an endangered species, would you want to rely on a technicians eyeball estimate of a match, or would you want some more accurate measurement In order to make the… Nt1310 Unit 1 Exercise 1 However, the DNA from the third to the seventh lane travelled down the gel as they were smaller due to fragmentation during amplification. If your experiment was unsuccessful, or marginally successful, offer plausible explanations for your results. This is very useful in the sense that no two restriction enzymes codes for exactly the same recognition site, giving it a unique characteristic that is specific for a strand of DNA. These proteins take in blue light and reflect green light, thus drastically changing the phenotype of the bacteria. A major factor in this experiment is the resistance to the antibiotic Ampicillin.

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Bacterial Transformation Lab Report

transformation of e coli with pgal lab report

Transformation of the pAMP to the E. The genes were successfully inserted, yielding a ampicillin resistant, fluorescent E. We took the LBAmp - petri dish into a dark closet to observe the bioluminescence of the colonies. Immediately after we reflamed the loop to sterilize it. Transformation with pGLO produces colonies that glow under UV radiation, rather than the blue colony phenotype produced by pGAL. The LB plate with amp plate negative control had no growth on it because the ampicillin killed all the E. We then used the inoculating tube to streak E.

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E. Coli Transformation Lab

transformation of e coli with pgal lab report

Materials and Methods: The materials needed in this experiment were an alcohol, starter plate of E. Mix by tapping c. Note that the loop was harshly spun in order to disperse the entire colony into the solution. The Negative Control a. While the alternative hypothesis is that the E. Figure one describes the results of the experiment and display it in a table.

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E coli Lab typemoon.org

transformation of e coli with pgal lab report

Within the plasmid, there are genes such as: the AraC protein which will regulate the Larabinose operons in E. Efficient Site Directed In vitro Mutagenesis Using Ampicillin Selection. A plasmid is a genetic structure in a cell that can replicate independently of chromosomes. That could have been due to the lack of precision while streaking, or perhaps there was no bacteria on our streaker to begin with. From the extent of the fragment one can figure the number of repeats by following equation. Explain the difference between each and why it is necessary to run each.

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Transformation of typemoon.org with pGal Flashcards

transformation of e coli with pgal lab report

The next step was to mix both tubes evenly and use pipettes to transfer 100µl of the transformation and control suspensions onto the corresponding nutrient agar plates. Then I would extract that gene, and insert bacteria into the pool of DNA, forcing transformation to occur. You will calculate the transformation efficiency from the data you collect from your experiment. The controls were chosen depending on which aspects of the experiment is emphasized. Typically, it is used Pglo Lab Report Abstract The purpose of this study was to see whether E. Experiment 1 Given: 0. Introduction to Bacteria The purpose of this experiment was to observe the transformation and growth of E.


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Transformation of typemoon.org with pGal (blue colony)

transformation of e coli with pgal lab report

Gently turn the tube upside down twice to mix and tape the tube so that all the liquid is in the bottom. Enzymatic cleavage of lactose by β-galactosidase Transformation by pGAL is also demonstrated by resistance to ampicillin. The null hypothesis is that the E. Bacterial samples are able to inherit new genes through three types of processes: transformation, transduction and conjugation. When the 90 seconds is up, immediately remove the E. Place the tube back on ice 3. What are some reasons why transformation may be unsuccessful? Place the tube back on ice.

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E. Coli Transformation Lab Report

transformation of e coli with pgal lab report

No IPTG used in this experiment. After the incubation, we heat shocked both tubes by transferring them from the ice directly into a 42 degrees Celsius water bath for 90 seconds, agitating the tubes during the water bath. It has its very own DNA that is different from the bacteria DNA. Using sterile technique, on the third day 2 15 mL sterile tubes, one with +plasmid and one with -plasmid. The ampicillin in the plate was eventually destroyed by the beta-lactamase that was produced by the successful transformants by their expression of the bla gene on pGLO 8 Transformation of Escherichia coli with Plasmid DNA pGAL Laboratory Protocol Rev. In essence, it is an indicator of how well the transformation experiment worked.

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lab report biology .docx

transformation of e coli with pgal lab report

Comparing the LB + to the LB - plate, the LB + had a thick lawn of bacteria colonies, with the LB - had nearly none present. Introduction Through this lab, we were able to learn how bacterial samples can inherit DNA and the process of transformation efficiency. As mentioned above, the methodology used was bacterial transformation protocol. Once in the chamber containing buffer solution and electrodes, the molecules of DNA will begin to migrate through the gel and form bands due to the negative charge of the phosphate groups in the backbone of DNA moving towards the positive electrode. Results Once experimentation was completed, we viewed our results Figures 1-4. Be careful not to scrape any agar off the plate, you just want the E. We use competent E.


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Lab Report 6: Transformation Lab

transformation of e coli with pgal lab report

In this process, the bacteria probably took in DNA from the dead cells, therefore transforming them into deadly cells. Assuming that the aliquot of pGLO that you used had a concentration of 0. They then proceeded to inject the animals with a virulent S and nonvirulent R strand. Calculate the efficiency for each of the four experiments below. Determine the mass of plasmid in the solution added to the agar plate 0.

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Pgal Transformation Complete Lab Revised

transformation of e coli with pgal lab report

Incubate cells on ice for a minimum of 30 min for up to 3 hr Day of the Lab before students arrive : Preparation of Aliquots of E. Squeeze and vigorously shake the plastic bottle to break up the solid agar into chunks d. Discuss the mechanisms of gene transfer using plasmid vectors. The plasmid pGAL contains the E. Solution in line 5 is treated with both SmaI and XhoI.

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