Pglo Lab Report

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.

The purpose of the PGLO lab was to be able to perform a procedure known as a genetic transformation. We used a procedure to transform bacteria with the gene that codes for a Green Fluorescent Protein (GFP). The actual source of the GFP gene that we used in this complicated experiment is the bioluminescent jellyfish Aequorea victoria. This protein causes the jellyfish to glow under a UV light that was provided in the dark. After the transformation procedure, the bacteria showed their newly acquired gene from a jellyfish and produced the fluorescent protein, which as a result, causes it to glow. If the bacteria glowed in the dark, that was the initial sign that the experiment was successful.

The hypothesis above tested the insertion of the pGlo gene to see if the bacteria, E.Coli, will reproduce and grow in the presence of ampicillin and to see if it will cause a green fluorescent glow. (PGLO™ Bacterial Transformation Kit,2017). Based upon the results from this experiment the hypothesis did support the hypothesis and that the presence of the pGlo gene inserted into the E.Coli did cause for growth and fora fluorescent glow to occur. In the experiment, the petri dishes that contained no pGlo (-pGlo) did not show any reproduction nor did a green glow appeared in both dishes. Unlike the two petri dishes, that contained the pGlo gene and ampicillin, the data data showed both reproduction and a glow in the petri dishes.

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

Genetic transformation is the change caused by genes. This transformation includes the insertion of a gene into an organism, changing one of the organism’s traits. There are many other uses for genetic transformation including the altering of plant genes coding for frostbite, pests and spoilage resistance. It can also be used to digest oil spills and even alter in gene therapy to transform sick cells into healthy ones. This particular experiment will include the transformation of the bacteria with the GFP ( Green

Next, each tube was opened and 250 µl of transformation solution was added to each and then placed in the ice. While the tubes were on the ice, a sterile loop was used to pick up to 2-4 large bacteria colonies of bacteria from the starter plates and immerse to both +pGLO. The tubes were then placed back on the ice. Once the colonies of bacteria were well placed in the +pGLO solution, the same process was repeated for the –pGLO tube using a new sterile loop.

This pBlu lab had for purpose to present the changes of the strain of E. coli bacteria due to new genetic information being introduced into the cell. In this experiment we are freezing and heat shocking the E. Coli bacteria that is then forced to take the plasmid DNA. The E. coli then transforms the pBLu plasmid, which carries the genes coding for two identifiable phenotypes. After following the Carolina Biological steps our lab worked well and we able to see some colonies of bacteria on the plates. The x-gal plate showed a significant amount of bacteria to confirm that the pBlu plasmid took over the E. coli strain.

The LB Plate acts as a control for the LB/AMP because it shows that the bacteria without the plasmid, but still went through the process, survived. The second plate, which contained LB/AMP, left no trace of bacteria behind. This is because the AMP is an antibiotic, which kills off the existing bacteria (LB). This plate also acts a control because without the plasmid, the bacteria can’t grow in the presents of Antibiotics.

Genetic Transformation of E. coli Using pGLO Plasmid Introduction The bacteria E. coli is a competent bacteria which has the ability to accept foreign pieces of DNA and express them in itself. In this lab will be testing the hypothesis that E. coli is competent and can express foreign DNA by depositing pGLO DNA, which was created from the same DNA that makes jellyfish fluorescent, into the E. coli to make the bacterium glow. We are also testing the hypothesis that the pGLO DNA can make the E. coli resistant to ampicillin.

This experiment focuses on genetic engineering and transformation of bacteria. The characteristics of bacteria are altered from an external source to allow them to express a new trait, in this case antibiotic resistance. In is experiment foreign DNA is inserted into Escherichia coli in order to alter its phenotype. The goal of the experiment is to transform E. coli with pGLO plasmid, which carries a gene for ampicillin resistance, and determine the transformation efficiency. The bacteria are transformed by a combination of calcium chloride and heat shock. When the bacteria are incubated on ice, the fluid cell membrane is slowed and then the heat shock

Title Genetically transferring the GFP (green fluorescent protein) gene into E. Coli via heat shock Introduction Genetic transformation is the process by which desirable genetic material is extracted from one organism and forcibly placed in a different organism’s genetic complex. By adding foreign genes of organisms into different organisms, the genetic diversity of organisms is increasing exponentially and genetic transformation is one, if not the best, way to produce new alterations of genes as well as improve the functionality of other, already existing, genes. Although there are many different ways by which genetic transformation can be done, heat shock transformation was the method used in this lab. Heat shock is used to increase the

After the groups have heat shocked their samples they plate the DNA onto four pertri dishes. The four petri dishes have three different forms of agar: the first with plain agar, the second which is used twice contains ampicillin, the final plate contains both ampicillin and isopropyl-ß-D-thiogalactopyranoside. The isopropyl-ß-D-thiogalactopyranoside induces gene expression. While the ampicillin is an antibiotic for bacterial infections. This is when the positive versus negative DNA comes into effect the +DNA has the plasmid that fights against the ampicillin which should allow it to grow.

On the selective media, Lysogeny broth with ampicillin, there should be growth on the transformed plate because the successfully

Hypothesis: If genetic transformation involves the insertion of a gene into an organism in order to change the organism’s trait where genes can be moved from one organism to another with the aid of a plasmid and pGLO plasmid encodes for a gene resistant to the antibiotic ampicillin as well as the gene GFP which allows the bacteria, E. coli, to express the glowing gene by producing the fluorescent protein if switched on after having undergone transformation by adding sugar arabinose – which is a nutrient for the cell -, then when the procedure of the pGLO transformation lab is completed as instructed in the student manual, the agar plate +pGLO Lb/ampicillin will produce colonies of bacteria that do not glow, -pGLO LB/ampicillin will not produce any bacteria, and –pGLO LB (constant) will produce a lawn of bacteria that does not glow (instead appearing as a

If Genetic transformation has the meaning of “change caused by genes” and involves the placing of a gene into an life form in order to modify the organisms characteristic; the progression of placing genes from one life form to a different is used to assist of a plasmid and the pGLO plasmid codes the gene used for GFP as well as the gene for resistance to ampicillin. It is used to manage the expression of the fluorescent protein; hence, the GFP gene is able to be switched on by adding the sugar arabinose to nutrient medium of the cell, then the bacteria will be able to glow a bright green underneath UV light when arabinose is within the nutrient agar medium. Hence, then when one micro test tube +pGLO and –pGLO are labeled and placed into a foam rack and the tubes are open and using a sterile pipet used to transfer 250 micro liters of transformation solution (CaCl2 ) in each tube, position the two tubes on ice, pick up 2-4 colonies of bacteria with a loop, submerge the loop into the +pGLO tube, repeat steps for –pGLO, put in to ice, and put plasmid DNA into the pGLO; after the pGLO’s need a heat shock by placing the cold tubes into the 42 degrees Celsius hot bath for 50 seconds and back into ice for 2 minutes, later insert the 250 micro liters of LB nutrients broth into the tube and then placing 100 micro liters into the 4 plates, each individual plate contains +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp or –pGLO LB). If bacteria that contains +pGLO plasmids is resistant to