Pglo Transformation Lab

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.

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.

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.

In this investigation pUC19 plasmids were used as the vector due to its small size of 2686bp, high uptake efficiency by the host and fast replication time. Important features of this plasmid include the origin of replication and multiple cloning sites (MCS). The origin of replication allows the plasmid to replicate inside the host bacterium. The MCS is located within the lacZ gene and contains unique sites for the Xbal & EcoRI restirction enzymes to cut and produce sticky ends for the CIH-1 gene to bind to. Furthermore, the pUC19 plasmid also contains an ampiccilin resistance gene so only transforemed E.coli are able to remain viable when spread on the agar plates that also has the addition of ampiccilin. The lacZ gene encodes the β-galactosidase enzyme which aids in indentifying the recombinant E.coli from the non recombinant cells (Coventry University 2016).

The experimental part of the lab consists of setting up the materials needed. A sample of E.coli and a solution of calcium chloride are first obtained and placed in different test tubes. 630µL of Calcium Chloride (CaCl2) are then removed from the test tube and inserted into the test tube containing E.coli cells (Alberte et al., 2012). The newly formed substance of Calcium Chloride and E.coli is then mixed and incubated in ice for 10 minutes, making the cells more competent. Two test tubes are obtained and labeled; the first test tube is labeled with pUC18 and the second one with “Lux” to represent the plasmids being used. These two test tubes are then incubated in ice. 3µl of the set plasmid are added to each of the two test tubes. The test tubes are tapped to guarantee the cells are well

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

1.2 ml of the bacterial cultures are taken to pellet the cells and remove supernatant while

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.

Transformation is the transfers of virulence from one cell to another, through the transferring of genetic material. It was originally postulated in 1928 through the works of Federick Griffith, a British microbiologist. Griffith observed that the mutant form, non-virulent form, of the bacteria Streptococcus Pnumoniae could be transformed into the normal, virulent form, when injected into mice along with heat killed normal forms. He concluded that somehow the information the dead virulent form had transformed the mutant form into a virulent form.

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 protein. Research scientists use Green Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC. Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).

coli grown on the petri dish containing LB broth, amp and arabinose (ara) that was also exposed to the pGLO plasmid glowed and grew and an average of 121 colonies. This was unsurprising, as the petri dish contained LB broth, with the necessary nutrients for E. coli to grow, this allowed several colonies of E. coli to grow. While amp was added, this strain of bacteria had been exposed to the pGLO plasmid, this means that the ampicillin gene was transferred into the bacterial genome. This allowed E. coli to grow, despite the quantities of ampicillin added to the petri dish. While E. coli was able to grow, it was also able to glow, the phenotype for the gene GFP. The GFP gene is part of an operon. The inducer for this particular operon is arabinose. It turns of the repressor and allows RNA polymerase to transcribe the GFP gene and produce the phenotype of glowing. This is why this particular group of bacteria was able to glow and none of the others

In this lab, we will perform genetic transformation to E. coli. Transformation is an insertion of a gene into an organism in order to change or add certain traits to the organism. Genetic transformation is widely used in the biotechnology field, for examples: In medicine, gene therapy involves transforming a sick person’s cell with healthy copies of the defective gene that causes the disease. In research, bacteria are transformed with genes encoding human proteins for Bio-manufacturing production or for further study of these proteins. Today’s lab, we are transforming bacteria with a gene that code for green fluorescent protein (GFP). This gene originally came from the bioluminescent jellyfish Aequorea Victoria. GFP is the

The bacteria that was used in the experiment was Escherichia coli which also has the ability to communicate through quorum sensing. Quorum sensing controls bioluminescent, antibiotic production, competence this better understand the bacteria that we were working with (Bassler, 2012). We wanted the bacteria to express antibiotic resistance, bioluminescent and growth all of that is control by quorum sensing which is a type of communication between the populations of the Escherichia coli. In addition, Escherichia coli is a gram negative bacteria which appear as purple when is stained. Gram negative have a thin layer of peptidoglycan but the outer membrane has porins which lets antibiotics such as ampicillin to move through the wall and the bacteria can become resistant to antibiotics. In the experiment, one of the way we made the Escherilia coli was make them competent to allow them the express and share the important information between themselves in the form of genes in the selected plasmid. Plasmids help the bacteria to transfer important date for its survival and its one of the features of the plasmids that is used in the transformation. The Escherilia coli first needed to become competent to take the DNA with the help of calcium chloride

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.