This lab works to develop the understanding of bacterial transformation through the integration of a plasmid into E-coli bacteria. Understanding of plasmids, GFP, expression of genes and bacterial candidates is used to formulate a lab which demonstrates a variety of factors associated with transformation efficiency. It was deduced that there are certain requirements present in the pGLO plasmid for full gene expression and that an increase in transformation solution positively impacts the results with an increase in growth. Introduction:
The pGLO lab is a lab that uses the theory of bacterial transformation to undergo its procedure. Bacterial Transformation is the insertion of foreign DNA into a cell thereby altering its DNA. When the gene is introduced (pGLO), the genetic information is absorbed into the plasmid of the bacterium. The DNA is then coded as if it were part of the original DNA. This new DNA is classified as recombinant DNA. The method used in this lab for bacterial transformation is a combination of a calcium chloride solution followed by a heat shock. The heat shock is then used to increase the permeability of the membranes present in the bacteria which allows for the easy introduction of the foreign DNA. Fredrick Griffith was the first person to attempt a bacterial transformation lab in 1928. He combined a pathogenic and non-pathogenic strand of pneumococcus bacteria that was treated with heat, he was able to develop a mixture that contained no virulent bacteria yet killed all of the subjects it was tested on. This was a great breakthrough in the field of biotechnology.
The bacteria chosen in this experiment is E-coli. Escherichia coli has become a "model organism" for studying many of life's essential processes. Due to its rapid growth rate, simple nutritional requirements, well established genetics and completed genomic sequence, more is now known about E. coli than any other living organism. It’s ability to reproduce so quickly makes it a great bacterium to use as you can leave it overnight and visible results can be seen the next day. It is currently the most widely-used organism in molecular genetics. E. coli has a cell division rate of about once every 30 minutes, enabling rapid adaptation to the environment.
The success of this lab also hinges on the presence of plasmids of DNA in the E-coli. A plasmid is an arrangement of DNA that takes a circular shape. Originally evolved from bacteria, plasmids are uncontrollably replicating and express many of the genes responsible for antibiotic resistance. Plasmids can also be modified to express proteins and consequently other characteristics. In this lab the pGLO plasmid is introduced to E-coli DNA to create the characteristics that are desirable in the host bacterium. Contained within the pGLO plasmid are three gene sets that code for different characteristics. The first of these genes is a Beta Lactamase gene. The presence of this gene in the DNA forces the bacterium to code for Beta Lactamase which helps the subject resistant to ampicillin. The second gene is the GFP gene. Extracted from Aequorea victori, a bioluminescent jellyfish, the Green Fluorescent Protein causes the E-coli colonies to glow in the presence of arabinose. The final component of the pGLO plasmid is the araC regulator protein.(2) This gene causes a protein to be made that regulates or controls transcription of the GFP.
In just three years, the green fluorescent protein (GFP) from the jellyfish stated above, has vaulted from the obscurity to become one of the most widely studied and exploited proteins biotechnology. It’s ability to create and highly visible internal fluorescence is both amazing and useful. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic abilities. GFP has become well known as marker of gene expression and protein targeting. Mutagenesis and...
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