The pGLO experiment is a lab activity that is commonly used in high school and college biology courses to demonstrate the principles of gene regulation and genetic engineering. In this experiment, students transform a bacterium called Escherichia coli (E. coli) with a plasmid that contains the gene for green fluorescent protein (GFP). The transformation is accomplished using a technique called electroporation, which involves subjecting the bacteria to an electric field to create temporary pores in the cell membrane through which the plasmid can enter.
The goal of the pGLO experiment is to demonstrate the concept of gene expression, which is the process by which a gene's instructions are used to produce a specific protein. In this case, the GFP gene on the plasmid codes for the production of GFP, which is a protein that fluoresces green when exposed to certain wavelengths of light. By introducing the GFP gene into the E. coli cells, students can observe the expression of the GFP protein and understand how genes are regulated in living organisms.
To perform the pGLO experiment, students first need to prepare the E. coli cells and the plasmid. This involves growing the E. coli cells in a nutrient-rich medium and purifying the plasmid using a process called DNA isolation. Once the E. coli cells and plasmid are prepared, the transformation can be performed using the electroporation technique.
After the transformation, the E. coli cells are plated on agar plates that contain the antibiotic ampicillin. The ampicillin is included in the agar to kill off any cells that did not take up the plasmid during the transformation process. Only cells that have successfully taken up the plasmid will be able to survive on the agar plates.
Once the transformed cells have had a chance to grow on the agar plates, students can observe whether the GFP gene has been expressed. This can be done by exposing the cells to UV light, which causes the GFP protein to fluoresce green. Students can compare the fluorescence of the transformed cells to that of control cells that have not been transformed to see the effects of the GFP gene on gene expression.
In addition to observing the expression of the GFP protein, students can also test for the presence of the GFP gene in the transformed cells. This can be done using a technique called polymerase chain reaction (PCR), which allows for the amplification and detection of specific DNA sequences. By using PCR to amplify the GFP gene in the transformed cells, students can confirm that the gene has been successfully introduced into the E. coli genome.
Overall, the pGLO experiment is a useful tool for demonstrating the principles of gene regulation and genetic engineering. By performing this experiment, students can gain a better understanding of how genes are expressed and regulated in living organisms and how genetic engineering can be used to introduce new traits into organisms.
A process called heat shock was then used to insert the plasmid into the E. Each MCFT was then labeled according to group and colony to not mix the cultures. The cell does not include any other organelles to confuse or distract the student. In this way, the gene of interest is cloned. This experiment is meant to prove that through genetic transfer using plasmid DNA, the E.
Like most bacteria, E. The mobilization of pSS-2 from onestrain of E. The possible bacteria inside my petri dish could be any of the following: Bacillus subtilis, Bacillus cereus, Mycobacterium species, Corynebacterium species, Lactobacillus, Staphylococcus aureus, Micrococcus luteus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pyogenes, and Streptococcus pneumoniae Hypothesis Unknown Lab Report Essay 408 Words 2 Pages The unknown bacteria was then tested on multiple selective and differential media. . The purpose of this lab was to perform a procedure known as genetic transformation which allowed us to genetically engineer E. Our results for this petri dish mean that the bacteria was not killed by the ampicillin.
As for the other three plates, they all grew the E. The third case is the effect on bacteria when no gene for pGLO is introduced, but LB and ampacillin is still introduced, The fourth case is the effect on bacteria when no gene for pGLO is introduced, but bacteria is still placed in a LB enriched environment. 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. Also close the - pGLO tube. Pick up the + pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Using aseptic technique, the bacterium 16A and 16B were inoculated into labeled broth test tubes.
Shimomura noted that when Aequorin and Ca 2 were combined, blue light was produced, but curiously, the Aequorea Victoria jellyfish emitted a green light. INTRODUCTION: Known for their graceful movements, tentacles, and stinging ability, jellyfish instill fear in ocean swimmers, are avoided and considered a loathsome aquatic creature. A liquid culture was then made using the bacteria covered toothpick and 4 mL of LB-K to isolate the DNA. This nitrogen base will match up to its complementary nitrogen base on the opposite strand, with a purine, either A or G, forming a hydrogen bond with a pyrimidine, either T or C, respectively these hydrogen bonds between the complementary nitrogen bases is what holds the two strands together. Lastly, the Citrate Slant is a green color and it was used as a differential test to examine enzymes.
Our expectations were both unsupported and confirmed. Unknown Bacteria 557 Words 3 Pages In the laboratory, identification of an unknown bacterium is often necessary. Day of lab 1. Actual: Observations of E. A variety of vectors have been created, each being suitable for a particular use. Additionally, the size of the TN5 transposon is large enough that it will functionally disrupt any gene or regulatory region where it inserts and change its function.
Because the plasmid and foreign DNA fragments were… Bacterial Transformation Using Pvib Transformation is the manipulation of a bacterial cell's DNA in order to alter the cell's genotype or phenotype by absorbing free DNA from its surroundings. The transformants were grown on the LB with the tetracycline antibiotic, and on the LB without the tetracycline. This is an example of how the GFP gene is regulated. Conditional on where the TN5 transposon was inserted into the functional area designated on the pGlo plasmid map, there will be different results. These were made in test tubes labeled with group number 2 and the colony number previously listed.
In this lab, we used a procedure called heat shock, accompanied by a bacterial plasmid vector, to transform bacteria with a gene that codes for GFP Green Fluorescent Protein. One beaker with ice water. For the results, TN5 insertion would affect growth if it inserts in pAmpR and AmpR like it did while affecting glow everywhere but pAmpR and AmpR and noncoding region. In the two plates containing the pGLO and ampicillin, one of which also containing the sugar arabinose, the E. This plate was allowed to incubate overnight, When the plate was revisited, and found that the colonies of bacteria were a green color. Genetic transformation, inserting the pGLO plasmid into E.
In an inducible operon system, adding an inducer, in this case the arabinose, would cause the repressor to become inactive, allowing the RNA Polymerase to go through and create the GFP through protein synthesis. There will be GFP expression if the transposon is inserted into the AmpR or P AmpR. We can thus conclude that bacteria can take in foreign DNA through the process of transformation and that this foreign DNA can fundamentally change the bacteria ex: making it glow. Using the lit tea light, like a bursen burner, I flamed the mouth of the S. Unknown Bacteria Lab Report 1034 Words 5 Pages Therefore, acid fat, lactose fermentation, mannitol fermentation were not needed to be performed, because they are selective to a specific thing. These all interacted through a process of random mutagenesis where the TN5 transposon inserts itself into a functional DNA region to generate random mutations.
Tap the closed tubes with your finger to mix. Next, we used two sterile loops to transfer E. It is necessary to obtain a gene to modify genetic material. The LB-K contained kanamycin, LB-KR contained arabinose and kanamycin, and LB-KRA contained ampicillin, arabinose, and kanamycin. After receiving an unknown mixture, the sample was streaked for isolation onto TSA, blood agar, and MacConkey plates. Intercellular Gene Transfer Lab Report 1100 Words 5 Pages The Mut section is expected to have no growth as mutants require the amino acids leucine and valine to grow which is not provided in the minimal medium.
