An Understanding Of Bacterial Transformation By Plasmid Dna

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.

134). They are loops of DNA that are separate from the chromosomal DNA and can self-replicate in a cell, found mostly in bacteria (Brown, 2011; Addgene, 2015). Lederberg and William Hayes discovered that plasmids were being transferred from one cell to another, not the chromosomal DNA (Brown, 2011, p. 135). This discovery lead to plasmids being an essential tool for scientists. Scientists can engineer plasmids to have specific genes to introduce into new cells (Brown, 2011, p. 134). On a plasmid loop there will be an origin of replication (ORI) and a multiple cloning site (MCS) where the gene of interest is inserted (Bio-Rad, 2015). This region has specific restriction enzyme recognition sites, which are cut by the enzymes to open up the DNA where the new gene will be inserted (Jove Science Education Database, 2015). Most plasmids will also contain an antibiotic resistance gene allowing cell survival in environments containing antibiotics (Jove Science Education Database, 2015).

Control plasmids lux and pUC18 were introduced into E. Coli through a process of transformation.

This experiment was designed to test and observe the transformation efficacy of the pUC18 and lux plasmids in making E. coli resistant to ampicillin. Both plasmids code for ampicillin resistance, however, the lux plasmid codes for a bioluminescence gene that is expressed if properly introduced into the bacteria’s genome. The E. coli cultures were mixed with a calcium chloride solution and then heat shocked, allowing the plasmids to enter the bacteria and assimilate into the bacterial DNA. The plasmids and the bacteria were then mixed in different test tubes and then evenly spread onto petri dishes using a bacterial spreader, heating the spreader between each sample to make sure there is no cross contamination. Each of the dishes was labeled and then incubated for a period of 24 hours. The results were rather odd because every single one of the samples grew. Several errors could have occurred here, cross contamination or possibly an error in preparation as every single sample in the class grew, meaning all samples of the bacteria transformed and became ampicillin resistant.

DNA. The isolated plasmids are ran on 40 ml 1% agarose gel at 110 volts with 1.2 μl 6X track

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.

The purpose of this experiment was to show the genetic transformation of E. coli bacteria with a plasmid that codes for Green Fluorescent Protein (GFP) and contains a gene regulatory system that confers ampicillin resistance. A plasmid is a genetic structure in a cell that can replicate independently of chromosomes. In this lab, the Green Fluorescent Protein, which is typically found in the bioluminescent jellyfish Aequorea Victoria, was cloned, purified, and moved from one organism to another with the use of pGlo plasmids. It was hypothesized that if bacteria that were transformed with +pGlo plasmids are given the gene for GFP, then transformed cell colonies

Purpose The purpose of this experiment is to demonstrate how genetic engineering works to manipulate the genetics to express certain traits. This can be seen with the insertion of the pGLO plasmid onto Escherichia coli. Introduction All of the things that is required to make a particular organism function properly, is called the genome. Eukaryotes have a genome that is composed of chromosomes, plasmids, and other specific organelles, such as mitochondria, chloroplasts, etc.

How does the addition of pGLO plasmid to a solution containing E. coli bacteria affect the growth and characteristics of the bacteria? Genetic transformation is the incorporation of foreign DNA into an organism to potentially change the organism’s trait. Plasmids are small circular DNA that replicate separately from the bacterial chromosome. In nature, these plasmids can be transferred between bacteria allowing for the sharing of beneficial genes. Due to this characteristic, plasmids allow for genetic manipulation and can be moved between bacteria easily. The pGLO plasmid utilized in this experiment encodes the gene for Green Fluorescent Protein (GFP), which under the right conditions can produce a glow. The gene regulation system present in the pGLO plasmid requires

-Plasmid is a small, circular, double-stranded DNA molecule that naturally exist in bacterial cells. The range of length are from hundreds base pairs to thousands base pairs. This is a great tool for clone, transfer, and manipulate genes.

Plasmids are small double stranded circular non chromosomal DNA molecules containing their own origin of replication. Hence, they are capable of replication independent of the chromosomal DNA in bacteria. Plasmids present in one or more copies per cell, can carry extra chromosomal DNA from one cell to another cell and serve as tools to clone and manipulate genes. Plasmids used exclusively for this purpose are known as vectors. The genes of interest can be inserted into these vector plasmids creating a recombinant plasmid. Recombinant plasmids can play a significant role in gene therapy, DNA vaccination, and drug delivery [Rapley, 2000].

Isolation of plasmid DNA from three cultures of E.coli using a method known as the alkaline lysis method.

Since bacteria are haploid, asexually reproducing organisms it is important for these organisms to be able to accept genetic variability into their genome. A process called transformation, which involves absorbing small segments of DNA from deceased organisms in the natural world, does this. Transformation can also be mimicked in the laboratory using plasmid. Plasmids are small segments of DNA that occur in bacteria that allow us to regulate if transformation was successful. We attempted transformation of E. coli cells using plasmid called pVIB, which allows for luminescence and resistance to the antibiotic ampicillin, from Vibrio fischeri, however, we did not achieve a successful transformation.

The domestication of animals 30,000 years ago was the precursor to Genetic Engineering. Starting with the dog we have since been able to introduce desirable traits in all organisms. With the discovery of plasmids in the late 60’s we have been able to take genetic engineering even further. Plasmids are small circular DNA molecules used to amplify and replicate a gene of interest. These minute molecules have the ability to replicate with the chromosome or independently, allowing them to have up to a 100 copies in one cell. Plasmids are important because of their characteristics to transfer genes that occur naturally within them or acting as a vector to introduce foreign DNA into a host cell. With the use of plasmids genetic engineers are able to use bacterial transformation to make medicines. Bacterial transformation is the process in which a bacterial cell takes up foreign DNA and incorporates this DNA into its own. With the use bacterial transformation this part of Genetic Engineering has become the most important and widely used technique, creating life saving antibiotics and medicines. E.coli is generally used in these procedures because of its ability to adapt and grow exponentially. Genetic Engineers can then use large bacterial colonies, Ampicillin, and X-gal to indicate if b-glactosidase is present along with identifying the recombinant and non-recombinant colonies. The transformed bacteria that contains ampicillin will spread and survive either turning white

Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial