The Transformation Of E. Coli

Abstract In this lab of transforming bacteria that was experiment today, I will be identifying the process of bacterial genetic transformation and how to calculate transformation efficiency. The samples that will be used in today’s bacteria will contain samples of E.coli sand inserted DNA plasmid into their genetic sequence. If done correctly the results will show a successful genotypic and phenotypic mutations, which will display fluorescent under ultra-violent lights or show signs to being resistant to ampicillin. This experiment was primarily for the purpose of growing E. Coli bacteria.

This lab is about moving genes from one thing to another using plasmids. Plasmid has the ability to replicate, so it replicates independently, and separately from the chromosomal DNA. Plasmid are one or more small piece of DNA and they enter cells as a double strand DNA. When they enter the cell as a doubke strand they do not invade he chromosomal DNA. We will also transform bacteria into GFP which is mainly from the jelly fish Aequorea Victoria. The GFP causes the the jelly fish to fluorescent and glow in the dark. After the transformation, bacteria starts to make the GFP which causes them to glow a green color under a ultraviolet light.

In preparing for the bacterial transformation, DNA plasmid is introduced into the E. coli cells that will express newly acquired genes. Two tubes were used and labeled both as +pGLO and -pGLO. A solution of (CaCl2) was transferred 250 µl onto the two tubes. The tubes were placed on the ice. A sterile loop was then used to gather a single colony of bacteria from a starter plate. Now, that both tubes contain bacteria they were placed on the ice for 10 minutes. Four agar plates were labeled as: +pGLO LB/amp, +pGLO LB/amp/ara, +pGLO LB, -PGLO LB/amp. Heat shock was used to transfer both the +pGLO and -pGLO, at exactly 42°C. Time was observed for 50 seconds and quickly return the tubes to the ice for another 2 minutes. As the tubes, cold down they

The purpose of this lab is to use genetic engineering to transform E. coli bacteria by inserting the plasmid pGLO, and to then see if the bacteria was transformed by using the antibiotic, ampicillin.

What was expected that the strains of E. coli that did not have a resistance to ampicillin would not grow. The transformed strain also changed to a blue color when the X-gal was present in plate. The transformed cells also grew because they were free of the ampicillin because they possessed the amp gene that they used as an shield against the ampicillin antibiotic. The transformed cell who turned blue did so because the gene converted the sugar to a blue color but also contained the amp gene to ensure that they grew even when the ampicillin was present. The growth of the colonies on the plates

To do a transformation one micro test tube was labeled +pGLO and the other was labeled –pGLO, using a transfer pipette 250 µL of transformation solution was added to each tube in the foam rack. The tubes were then placed on ice for three minutes. During these three minutes, a sterile loop was used to pick up a single colony of E. Coli from the starter plate by gently running the loop over the agar. This loop was then inserted into the +pGLO tube and the loop was spun until the entire colony dispersed. Using a different sterile loop, the same procedure was used for the –pGLO tube. After both tubes had their own colony of E. Coli, they were placed on ice for another three minutes. DNA plasmid was added to the +pGLO tube by taking a new sterile loop and immersing it into the stock tube creating a film across the loop then inserting

The transformed bacteria showed growth despite the presence of ampicillin (Table 1), whereas the control plate with ampicillin did not show any growth, and the control plate with only LB agar showed the formation of a lawn of bacteria (Table 2). The transformed bacteria on the plate with LB, ampicillin and arabinose differed from the transformed plate without arabinose in that they glowed green under UV light. The bacteria without arabinose maintained an unaltered appearance under UV light. The transformation efficiency for the transformed bacteria was 5.2 × 104 transformants per microgram of DNA.

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.

Our research on recombinant DNA mainly consisted of two experiments: Transformation and gel electrophoresis. In our first experiment, four microfuge tubes were given to us: pKAN DNA, pAMP DNA, unknown DNA, and a TE buffer without DNA. The two positive controls, pKAN and pAMP, consisted of an antibiotic resistance gene respectively to their name. The pKAN plasmid contained the gene resistance for kanamycin while pAMP carried the gene resistance for ampicillin. The negative control, TE, only contained buffer without DNA. The fourth tube was our unknown plasmid, which was either pKAN or pAMP; and by way of artificial transformation, we would be able to initiate the identification of our unknown plasmid.

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

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

To start this laboratory, it must first label two transformation tubes of different color; one with – DNA and the other with + DNA. Then, using a P-1000 micropipette adds 250 ul of the ice cold transformation solution (CaCl2) into each tube and places both tubes on ice. Using a disposable sterile inoculating loop to transfer colonies of E. coli bacteria from the starter plate to the +DNA tube. Immerse the loop in the CaCl2 solution in the transformation tube and spin the loop until all bacteria is incorporated into the transformation solution. Repeat this step with the – DNA tube. Using a P-10 pipette, transfer 10 ul of pGLO directly into the + DNA tube. Tap the tube lightly with a finger to mix. Place both tubes onto ice, and incubate for 10 minutes.

Tiffany O’Connor                                                         November 9th, 2017              Plasmid Transformation      Purpose: Plasmid transformation is the process of transferring foreign exogenous DNA into a host cell to change its phenotype.   Theory and Background: Plasma transformation can occur naturally when a cell alters its genetics by taking in DNA from its environment. Genetic transformation can also be induced in a laboratory setting, called artificial transformation. Artificial transformation is accomplished by choosing a competent bacterium and introducing a plasmid.   E. Coli is the bacteria used in this lab.

This experiment was performed to test the hypothesis if LB nutrient broth, +pGLO and -pGLO Ampicillin, and Arabinose was placed in the E. coli plates, then there will be a significant growth in the newly transformed bacteria and it will possess the ability to glow under UV light. The measurements were recorded from the bent glass tube in each glass test tube. The transformation protocol tested for the newly possessed traits in E.coli bacteria. Throughout the experiment there were many probable reasons for failure. If the pipettes and sterile loop were not thrown out in between each use, a cross contamination could cause a miscalculation in the experiment causing the data results to fail. The hypothesis that was tested was validated due to the positive results with each experiment stating that newly transformed organisms due in fact pass on traits.

The transformation began with the plating of bacteria on agar plates, one without antibiotic and the other with a suitable antibiotic. After one night incubation of bacteria at 37 °C, the number of colonies multiplied were counted and the amount of plasmid transformed was estimated. The details of the procedures can be found in the handbook ( De Smit &