Genetic Transformation Lab

Michelle Trujillo 5702361 Michaela Salisbury BSC 1010L U60 Effects of pUC18 and lux Plasmids on Ampicillin Resistance of Escherichia coli Abstract This experiment was designed to test and observe the transformation efficacy of the pUC18 and lux plasmids in making E. coli resistant to ampicillin. Both plasmids code for ampicillin resistance, however, the lux plasmid codes for a bioluminescence gene that is expressed if properly introduced into the bacteria’s genome. The E. coli cultures were mixed with a calcium chloride solution and then heat shocked, allowing the plasmids to enter the bacteria and assimilate into the bacterial DNA. The plasmids and the bacteria were then mixed in different test tubes and then evenly spread onto petri dishes using a bacterial spreader, heating the spreader between each sample to make sure there is no cross contamination. Each of the dishes was labeled and then incubated for a period of 24 hours. The results were rather odd because every single one of the samples grew. Several errors could have occurred here, cross contamination or possibly an error in preparation as every single sample in the class grew, meaning all samples of the bacteria transformed and became ampicillin resistant.

Abstract The pGLO plasmid is engineered to express green fluorescent protein (GFP) in the presence of the sugar arabinose as well as the ampicillin resistance gene β-lactamase (bla) (Brown, 2011). Original E. coli HB101 do not have ampicillin resistance or the GFP gene allowing them to glow under UV light. In this experiment, we transformed E. coli HB101 with the pGLO plasmid by heat shock to make the bacterial cells competent, allowing the plasmid to enter the cell (Brown, 2011). The mixture of bacteria with pGLO plasmid were given recovery time after heat shock, then spread on LB/amp and LB/amp/ara agar plates. The bacteria mixture with no plasmid added were spread on LB and LB/amp agar plates and all four plates were incubated at 37°C for

Introduction: Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial

In the pGLO Bacterial Transformation lab, Escherichia coli is transformed with a gene encoding green fluorescent protein by inserting a plasmid containing the GFP gene, beta-lactamase, and arabinose into the bacterium. Successfully transformed bacteria will grow in the presence of ampicillin and glow a bright green color under ultraviolet light. The sugar arabinose is responsible for switching on the GFP gene in the transformed cells, without it, the gene will not be expressed.

The purpose of this experiment is to make E.Coli competent so that it can be transformed in order to become immune to ampicillin, then we would be able to determine the transformation efficiency of the culture. We determine this by preparing 4 plates of E.coli, each labeled “LB-plasmid”, “LB+plasmid”, “LB?Amp-plasmid”, and “LB/Amp+plasmid”. This meant that either should have lacked plasmid and Ampicillin, with plasmid but lacked Ampicillin, without plasmid but with Ampicillin, or were with Ampicillin and plasmid, respectively. Then we made the bacterial cells competent by adding CaCl2 to 2 vials of the colony (one with plasmids), and incubating on ice, then heat shocking, and returning to ice. Luria Broth is then added and left to sit for 5-15

How does the addition of pGLO plasmid to a solution containing E. coli bacteria affect the growth and characteristics of the bacteria? Genetic transformation is the incorporation of foreign DNA into an organism to potentially change the organism’s trait. Plasmids are small circular DNA that replicate separately from the bacterial chromosome. In nature, these plasmids can be transferred between bacteria allowing for the sharing of beneficial genes. Due to this characteristic, plasmids allow for genetic manipulation and can be moved between bacteria easily. The pGLO plasmid utilized in this experiment encodes the gene for Green Fluorescent Protein (GFP), which under the right conditions can produce a glow. The gene regulation system present in the pGLO plasmid requires

In our hypothesis we stated that only the container containing all of the components +pGLO, LB broth, ampicillin, and arabinose would be the one that genetically transformed. In order for the bacteria to grow at a rapid pace all it needed was LB broth but when you added ampicillin, an antibiotic, it killed off all of the bacteria. +pGLO has the gene to resist the antibiotic so when that was added it was allowed to grow but there was no sugar to turn on the glowing protein. Finally, after arabinose, a sugar, was added it turned on the switch located in the +pGLO for the fluorescence and enabled to grow and glow.

Abigail Soberano Zapata Life 102 Lab L13 Lab Report The effects of green fluorescent protein on E. coli bacteria Introduction: Genetic transformation occurs when genes are inserted into another gene to change the organism’s trait (Weedman2016). In this experiment, we proceeded to transform the E. coli bacteria with a gene that contained green fluorescent protein. The green fluorescent protein is used in experiments because it beams a green color under a UV light (Chalfie2008). Typically, it is used to mark the expression of genes, which is why it serves as the symbol for all gene expressions (Tsien1998). In the experiment, we will be using pGLO as the organisms that will transmit the disease, otherwise known as a vector. The pGLO in the experiment

Abstract This experiment was performed to assess the efficacy of genetic transformations on bacteria via plasmid DNA coding for ampicillin resistance and green fluorescent protein. Genetic transformation was studied by taking transformed and untransformed Escherichia Coli (E. coli) and placing them on various media to observe gene expression via growth and color under UV light. The transformed E. coli were able to grow on ampicillin while the untransformed E. coli, which lacked the plasmid genes for ampicillin resistance, only grew on nutrient broth. In the presence of arabinose, the transformed E. coli glowed green. These results support the previous scientific understanding of bacterial competency, vectors, and gene expression and support gene transformations as an effective method to transfer the desirable DNA of one organism into another organism’s DNA. These results can be applied to real world issues such as medical treatments, food production, and environmental conservation.

Hypothesis: If a gene that codes for Green Fluorescent Protein transforms bacteria and GFP glows when transformation occurs, then when two micro test tubes have 250 microliters of transformation solution and places in an ice bath, then 2-4 bacteria colonies are added to each tube with a sterile loop; then a plasmid (pGLO) is added to one of the tubes, incubated in ice for 10 minutes, then heat shocked for 50 seconds at 42 degrees Celsius, then back into 9ice for two minutes; then LB nutrient broth is added to both tubes (250 microliters) and set out at room temperature for 10 minutes. Then, 100 microliters of each solution in the tube are added to four

Genetic transformation occurs when an organism’s genetic makeup is altered due to the introduction of new genetic information which is then incorporated into the organism’s genome. In this lab the pGLO plasmid is introduced into E. Coli bacteria, and incorporates the genes which code for the GFP and beta lactamase to the bacteria’s genome which as a result will be modified. To test the effects of the plasmid, bacteria treated with the plasmid were grown on separate plates, the first containing LB nutrient broth and ampicillin, another containing LB nutrient broth and arabinose and another containing LB nutrient broth, ampicillin and arabinose. Two more plates were grown, one with LB nutrient broth and ampicillin and the other with only the LB broth, using cells that did not contain the plasmid. Since the lab was about genetic transformation, the goal was to find which plate would glow. It was found that the plates that were not exposed to the plasmid did not glow, and the plates containing LB and arabinose and LB, ampicillin and arabinose did glow. The plates containing ampicillin, the antibiotic that kills E. coli did not grow whereas the remaining plates at least had some growth.

Title: Purification of Green Fluorescent Protein Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria. Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel electrophoresis is used to check and analyze the pure

The goal of this experiment was to create a “super e. coli,” as mentioned in the introduction, by co-transforming the pKan and pGlo plasmids. Since the e. coli were not able to absorb both plasmids at the same time, it can be noted that the transformation did not work. The plates containing the pKan and pGlo plasmids separately demonstrated a successful transformation, indicating the bacteria’s inability to take in two plasmids at the same time. Both of these plates acted as our controls, so in the end we could

The incorporation a plasmid vector, pGLO, into Escherichia coli via transformation is described. This recombinant plasmid that has been altered contains the ability to fluoresce green under ultraviolet light because of the Green Fluorescent Protein (GFP). pGLO also encompasses an antibiotic resistance gene, bla, that allows E. coli to grow in media that contains penicillin derivative antibiotics such as ampicillin. We conclude that the protein, GFP, is indeed responsible for the green fluorescence as pGLO contains the gene to encode for it, which is turned on when arabinose is present. Additionally, when the protein is subjected to denaturation, it decreases the fluorescence and changes the conformation of the protein structure.

PROCESSES USED IN GENETIC ENGINEERING APPLICATIONS The field of genetic engineering is comprised of multiple different subsets. Some of the specific research subsets of the field include gene therapy, genetic modification, genetic enhancement, and cloning. Gene therapy and genetic enhancement use similar processes to alter specific portions of the DNA, whereas cloning has a more specific method which is different in relation. The basic process of genetic modification will be used as an example to illustrate the technical aspects of research areas like gene therapy and genetic enhancement. In addition to genetic modification techniques, cloning has specific applications in animals. A specific method of cloning, somatic cell nuclear transfer, will be discussed in addition to genetic modification to demonstrate the technical skill associated with this application of genetic engineering.