Biological Transformation Of Bacteria And Pglo Plasmid Dna

In this lab, the organism that we have been working with is the bacterium, Serratia marcescens. S. marcescens is a member of the Enterobacteriaceae family, and tends to grow in damp environments. S. marcescens is an ideal bacterium to work with in the lab because it reproduces quicker than other bacterium. This bacterium produces a special pigment called prodigiosin, which is red in color. The prodigiosin pigment is intensified when S. marcescens is grown at higher densities. During our experiment, temperature, pH, salinity concentration and oxygen requirements were tested on S. marcescens to measure their optimal growth and prodigiosin production.

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

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 plasmid pGLO contains an antibiotic-resistance gene, ampR, and the GFP gene is regulated by the control region of the ara operon. Ampicillin is an antibiotic that kills E. coli, so if E. coli, so if E. coli cells contain the ampicillin-resistance gene, the cells can survive exposure to ampicillin since the ampicillin-resistance gene encodes an enzyme that inactivates the antibiotic. Thus, transformed E. coli cells containing ampicillin-resistance plasmids can easily be selected simply growing the bacteria in the presence of ampicillin-only the transformed cells survive. The ara control region regulates GFP expression by the addition of arabinose, so the GFP gene can be turned on and

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.

Chemotaxis (chemical signal) and phototaxis (light stimulus) stimulate the flagellation to rotate counterclockwise (run) or clockwise (tumble).

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 do you insert the plasmid inside the bacteria? What process do you use? (1/2 pt)

Finally, our results showed that the bacteria on the +pGLO plate with the amp and ara nutrients had the glowing bacteria. Even though the plate had only one colony of bacteria, it was the only one that glowed. This proves our hypothesis to be partially true because it shows how we were right in choosing the +pGLO plate with all the nutrients to have the glowing bacteria. In conclusion, our hypothesis was half right and half wrong, because the +pGLO with all the nutrients had the only glowing bacteria, but didn;t have the most colonies of bacteria like we first

In two weeks of genetic transformation, we were able to successfully complete the objectives of the lab by correctly performing a transformation of bacterial cells. During the lab, we were able to successfully complete the components that were necessary for a transformation of bacterial cells. Viable cells were transformed, able to grow, and were put in a sterile environment conducive to growth. With the exception of the plate labeled LB/Amp: -pGLO, our results followed the lines of a successful experiment. The LB/Amp: -pGLO plate was contaminated and had colony growth in it. In order for the transformation to be completely successful the LB/Amp: -pGLO plate would have had zero colony growth. As stated in the laboratory manual, biotechnologists

Prediction: If UV light mutates the DNA of Serratia Marcescens then the red pigment colonies of the bacteria will no longer be produced.

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

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.

Emerge of recombinant DNA technology provided an immense potential in the field of plant transformation. Transgenic plants detection in most crop species in order to minimize regeneration of non-transformed tissues after transformation requires the use of selectable marker genes and selective agents. The commonly used selectable markers in plant transformation systems are genes conferring resistance to toxic compounds such as herbicides or antibiotics. The negative selectable marker genes routinely used in Nicotiana tabacum transformation are genes that confer resistance to the antibiotic kanamycin. However, the presence of these genes or the derived proteins are undesirable in crop plants grown in the field, because of the public concern

DNA encodes the genetic instructions for cells to carry out their daily activities. DNA can come in many forms; plasmids for example are small circular DNA molecules found in most bacterial cells. Though plasmids may not be essential for the life of bacteria, it can give cells resistance in foreign environments. For the purpose of this experiment, an ampicillin-resistant plasmid is introduced to E. coli. This is done through a process of genetic engineering called transformation. Transformation works through the uptake, incorporation, and expression of a foreign gene to alter the genetic code of a cell. Three conditions are needed for successful transformation: a host, a vector, and a technique to identify the transformed cells. E. coli is used in this experiment as the host (E. coli is commonly used in biotechnology due to its rapid rate of growth and short reproduction time). A vector mediates the transfer of foreign DNA into the host cell. Plasmids are commonly used vectors that will also be used in this experiment. The procedure of tagging is used in this experiment to differentiate the transformed cells from those that were not. The learning objectives of this experiment are to: observe the process of bacterial transformation in an experiment; and demonstrate a change in phenotype due to uptake and expression of the genes in a known plasmid.