The Effects of Using the Heat Shock Treatment to Deliver a Vector in Genetic Transformation Introduction:
In this experiment we are testing what is required for E. coli to successfully grow on LB (Luria Broth) plates with ampicillin and determining if any genetic transformation has occurred. By combining +pGLO LB and ampicillin we should get an ampicillin resistant gene and by using –pGLO we should create a non-genetic resistant bacteria. The pGLO plasmid has the GFP (green fluorescent protein) gene and the gene that allows the plasmid to be resistant to the antibiotic ampicillin. The most important part of this experiment is the “heat shock treatment” because the E. coli membrane becomes permeable and increases the competency of the cells which in the end enhances the rate of successful transfer.
According to Weedman (2009) genetic transformation is using foreign DNA and inserting it into an organism is known as “change in genes.” This is common in biotechnology in the agricultural sector, bioremediation, and in the field of medicine. In agricultural sector, genes can be genetically changed into plant species that originally lacked that trait. In bioremediation, a bacterium is altered with genes that allow them to digest oil spills. Furthermore in medicine, diseases can be treated with healthy copies of the defective genes which could save the individual.
Weedman (2009) also states three different ways that genetic transformation can occur. Those include projective bombardment, electroporation, and heat shock. In projectile bombardment cells are attacked with tungsten pellets coated with foreign strands of DNA with a gene gun. In electroporation cells are bathed in a liquid that contains foreign DNA and is given a series of electrical pulses which increases the permeability of the cell membrane and causes the cells to take up the DNA from the surrounding medium. Heat shock does pretty much the same thing as electroporation except that the cells are subjected to a sudden increase of temperature. To transfer the gene from one organism to another a vector is used. Two types of vectors are phages and plasmids. Phages are viruses that affect the bacteria. This can be used to infect a bacterial colony which injects genetic material. A plasmid is a ring of DNA that contains genes for traits that are beneficial to the bacterial cell. In the environment, bacterial genes assist in the adaption of bacteria in new environments. The occurrence of resistance to antibiotics is caused by the transfer of plasmids, which contain genes for antibiotic resistance among bacterial strains. There become multiple strains of bacteria that develop immunity to the medication.
Zhou (2010) researched human papillomavius (HPV) which is a virus that induces cancer and premalignant diseases with an attempt to find a gene vaccine that would help cure HPV. He used a phage vector that would encode the viral E7 oncoprotiens as the tumor antigens from various HPV serotypes. To minimize the risk of cell transformation and improve immune responses he genetically inactivated E7 proteins using heat shock. The fusion of the protein gene was packaged in a phage vector efficient in vivo gene expression. The results of the experiment concluded that after an intramuscular injection, the AAV1 vector produced stronger HPV-specific cytotoxic responses and interferon-gamma secretion compared to the AAV2 vector. Prophylactic immunization with AAV1 protected 100% of the mice from tumor growth of over a year compared to the control mice that were vaccinated with either the LacZ vector or saline these mice grew large tumors, which then resulted in them dying within 6 weeks after vaccination of atumorcilline. The single dose of AAV1 completely protected the mice against second and third challenges with higher numbers of TC-1 cells. In addition to lower responses against the E7 antigens, AAV2 vector prophylactic immunization was also enough to protect 100% of the...
Cited: Kikkert, R. J. "Biological Projectiles (phage, Yeast, Bacteria) for Genetic Transformation of Plants."
REFDOC. Web. 04 Nov. 2010. <http://cat.inist.fr/?aModele=afficheN&cpsidt=1703156>.
Zhang, G. "Enhancing Electro-transformation Competency of Recalcitrant Bacillus
Amyloliquefaciens by Combining Cell-wall Weakening and Cell-membrane Fluidity
Disturbing." Analytical Biochemistry (2010). Print.
Zhou, L. "Long-term Protection against Human Papillomavirus E7-positive Tumor by a Single Vaccination
of Adeno-associated Virus Vectors Encoding a Fusion Protein of Inactivated E7 of Human
Papillomavirus 16/18 and Heat Shock Protein 70." Human Gene Therapy 21.1 (2010): 109-19. Print.
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