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Escherichia Coli Absorbancy Of Green Florescent Protein Through Heat Shock

1459 words - 6 pages

___________Escherichia Coli Absorbancy of Green Florescent Protein through Heat ShockIntroductionThe absorbance of plasmids by bacteria is one of the characteristics that make bacteria able to change characteristics. The manipulation of bacteria, as used for the formation of insulin, is done on the large part by the introduction of plasmids in the bacteria's environment. However, simply introduction of the plasmid in the bacteria environment is not enough to cause a necessary mutation. Among other techniques, heat shock can be used to help the acceptance of the plasmid by the bacteria. The amount of Escherichia Coli (Dh5α) transformed with the Green Florescent Protein Plasmid (pGFPuv) is quite low with the current temperature. The heat shock temperature used could be optimized to create a better acceptance rate of the plasmid.There are many different strains of GFP. The original GFP was derived from a complementary DNA of the Aequorea victoria. The protein expresses a fluorescent product when in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Other substrates or cofactors are not needed to fluoresce, so the GFP is quite versatile. It also can be used to monitor the gene expression or protein localization in living organisms without damage to the cells. (Chalfie 1995) When pGFPuv is expressed in E. Coli it fluoresces when exposed to the ultraviolet light spectrum. Therefore a successful transformation can be detected by luminescence with exposure to ultraviolet light.In order for a transformation, pGFPuv must be taken in by the E. Coli cells, and the cells must recover from the heat shock procedure. The procedure increases the permeability of the cell membrane by rapidly changing the temperature of the environment of the bacteria. This temperature change also can denature the cell proteins and other key parts. With the external stress on the cell, heat shock proteins, a group of proteins activated when subject to external stress, come into play. These proteins are produced normally in cellular processes but stay dormant until their function is necessary. The heat-shock proteins are divided into three catagories: effector molecules, a signal for heat shock protein synthesis; proteases, which degrade foreign proteins; and chaperones, which help protein folding processes (Cha 1999). Under normal conditions, the plasmid would not be able to penetrate the cell membrane. However, with heat shock, the cell membrane becomes permeable because of the increase in pore size. For this reason, the temperature of heat shock should be optimal to increase permeability of the pores but not too great as to denature the cell membrane and proteins.At present acceptance of the GFP plasmid by E. Coli is quite low. There exists a variance between different plasmids and bacteria and the optimal temperature for heat shock. (Weber-Ban 1999, Tavernarakis 2000). The temperature varies on the size and mass of the plasmid as well as the type...

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