Transformation Of Recombinant Egfp / Coli And Analysis With Biotechnology And Bioinformatics Tools

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.

coli the bacteria that will be used to house the GFP is the competent cell. They are called competent cells because they are able to accept foreign DNA or plasmids. They are first observed in 1928 by Frederick Griffith where he proposed that bacteria are capable transferring genetic information (Havarstein, 2010). These cells such as E. coli are normally made competent by exposure to a rich calcium environment, where the mixture calcium chloride will be used in. Calcium chloride’s positive charges will cancel out the negative charge of the plasmid and E. coli’s cell wall weakening it, making it easy to pass.

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

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

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

Lab Report on pGAL Transformation In order to understand this lab the student first needs to understand how recombinant DNA is formed. To begin, the student extracts a plasmid, which is a circular strand of DNA found within bacterial cells, from the bacteria. Restriction enzymes begin to cut the plasmid at certain sequences of nitrogenous bases.

Escherichia Coli is a rod-shaped bacterium that measures approximately 0.5 μm in width by 2 μm in length. It is a Gram-negative bacterium. Its cells stain gram negative because they have a thin cell wall with only one to two layers of peptidoglycan. They live in environments with higher temperatures rather than cooler temperatures. E. coli is said to be the “model organism”. Many microbiologists use these bacteria as a resource for understanding other prokaryotic life and are the most carefully studied life form on the planet. It was discovered by German-Austrian pediatrician Theodor Escherich in 1885.

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].

Escherichia Coli (E-Coli) is a member of the Bacteria kingdom. E-Coli is a unicellular microorganism (“Where”). This bacteria lives in human and animal intestines. It has been known to cause a number of food illnesses (LeClerc 1996). To be able to survive in humans and animals, this bacteria makes a protein that is resistant to the acidic PH in the intestines (Winfield). In the right conditions, such as a warm, nutrient rich environment, it can split and replicate in about 20 minutes (Berg 2004). Since it grows quickly, it is a common bacteria used in research (Don 2009).

A transformation was performed so a vast quantity of the recombinant plasmid DNA might be obtained. There was a total of six transformations performed from four DNA ligations (excluding the fifth ligation). All six transformations were then incubated on ice for five minutes, followed by a heat shock, which came from putting the transformations in a 42-degree Celsius heat block for two minutes. Afterwards, the transformations were incubated on ice for another two minutes, then placed in a 37-degree Celsius incubator overnight, then plated. All the transformations included E.coli and luria broth, but differed in their other growth mediums. The first transformation consisted of the 1:1 molar ratio of pET-41a(+) to egfp ligation, kanamycin and

Bacteria play major role on production of proteins especially for proteins that are required to be synthesised in glycosylated, bacteria have outstanding appearance system and also their relative simplicity biologically. Bacterial processes also be likely to be cheaper than eukaryotic cell processes because of lesser media costs and shorter method. The most ordinarily use of bacterium for recombinant protein production is Escherichia coli, an enteric bacterium that has a long pedigree of safe use in laboratories and industry .E. coli is a mainly suitable host because it is well categorized physiologically and metabolically, it was among the first organisms to have its entire genome sequenced and many molecular biology tools a for engineering DNA sequence to generate functionally. E. coli is

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