Pglo Transformation Lab Write Up

Introduction In molecular biology, transformation is the genetic alteration of a cell resulting from the uptake and expression of foreign genetic material (DNA) [1]. Bacteria evolve rapidly not only by mutation

Purpose: The purpose of this experiment is to teach the students step by step on how the genetic transformation process works using pGLO. Genetic transformation is when an individual cell’s genetic material is changed by exogenous DNA.

The objective of this experiment was to genetically transform E.coli cells to express ampicillin resistance and to produce the green fluorescent protein by using the pGLO plasmid. It was hypothesized that only the cells with pGLO DNA added to the solution would be able to survive in the same environment as the ampicillin, and that only the cells grown in the plate with arabinose would fluoresce bright green. The results supported the hypothesis, showing that the +pGLO LB/amp/ara plate was the only plate that had fluorescent cells and grew. Additionally, that +pGLO cells were the only ones that grew on an LB/amp plate.

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

After that, a new sterile loop was used to immerse the pGLO plasmid DNA stock tube into the +pGLO tube but no into the – pGLO tube. Both tubes were well closed and put back on the ice for 10 minutes. While both tubes were sitting on ice, the four plates were labeled. LB/amp plate was labeled +pGLO, LB/amp/ara plates were labeled +pGLO, LB/amp plate was labeled – pGLO, and LB plate was labeled – pGLO. After that, the tubes were transferred into a water bath set at 42° C for exactly 50 seconds, then placed rapidly back on the ice for another two

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.

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.

The field of biotechnology involves the concept of genetic engineering, altering the DNA/genetic material of an organism using information from a different one. The process in which bacteria can obtain this manipulated genetic information from another source is called genetic transformation. The goal of this experiment was to genetically transform Escherichia coli bacteria’s DNA by inserting the vector pGLO plasmid which codes for ampicillin resistance as well as the green fluorescent protein, GFP. For the experiment, the E. coli bacteria were separated into two groups; control and

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.

Next the tubes were placed in an ice bath, while obtaining a sterile loop to swipe a single colony of E.coli to put into the tube. After gently swiping a colony onto the loop, it was then spun in the +pGLO tube to get it to come off, returned to the ice bath. Next using a different sterile loop, it was swooped it in a container labeled pGLO plasmid DNA and again spun it ONLY into the tube with the solution labeled +pGLO to get it to come off. After about 10 minutes on ice the tubes were then placed into a 42ºC water bath for 50 seconds exactly, and immediately after placed them back into the ice bath. Finally, after 2 more minutes in the ice bath the tubes were separated into 4 containers. 250 ul of +pGLO solution was added to the containers containing +pGLO, LB broth, with ampicillin and +pGLO, LB broth, ampicillin, with arabinose. Also 250 ul of the –pGLO solution was added to the 2 containers containing LB broth, and ampicillin, with LB broth. Using a sterile loop for each plate the solutions were spread out gently and thoroughly on to the containers with agar. After the containers were incubated in 37ºC for at least 24 hours, the results were observed and disposed of (Weedman,

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

Introduction Genetic transformation is known to be the conversion of a genotype into another with the addition of DNA from an external source. Frederick Griffith was the first person to use genetic transformation in 1928 with Streptococcus pneumoniae (Griffiths et al. 2000). There are three different methods that are used in genetic transformation to get the vector DNA into the nucleus of the competent cell. The methods are, projectile bombardment, electroporation, and heat shock. Heat shock is the most common method is projectile bombardment because it guarantees the mixture of the two DNA (APSnet…2014).

Transformation is any change in an organism that alters its general character. It means “change caused by gene.” (biology-online.org) Genetic engineering is the process of manually adding new DNA to an organism. The main goal of genetic engineering is to add one or more new traits that the organism does not already have. ( ) There are many benefits of genetic engineering. With genetic engineering many illnesses and diseases can easily be prevented by isolating the gene that causes them. Also, genetic engineering has the ability to increase genetic diversity and pinpoint desirable traits of certain organisms. There are also various disadvantages of genetic engineering. For example, the process of genetic engineering is a long process that is risky and tricky. You need a variety of information in order to attempt the process. There is also debate about genetic engineering. Some people believe that it messes with the moral issues in religion.( )

In 1928, Fred Griffith first discovered genetic transformation by infecting mice with unencapsulated and non-pathogenic pneumococci (Lacks 2003). This was the start that opened up the field of biotechnology. Genetic transformation is a process where foreign DNA crosses a membrane of another cell and then alters the genetic material (Encyclopædia Britannica 2015). Genetic transformation can occur in a few different ways: projectile bombardment, electroporation, and heat shock (Weedman 2015). Heat shock is defined by increasing the temperature of cells environment making the plasma membrane become more permeability allowing new DNA to transfer into the cell (Weedman 2015). The cell receiving the new DNA, also known as competent cells, can either amplify the DNA or clone the DNA (JoVE 2015). The most common type of DNA used to perform genetic transformations is plasmids: small, round DNA molecules that still contains two strands of DNA that has the ability