Some bacteria are able to go through transformation making new combinations of genes. Transformation is a way of gene variability in bacteria. This experiment is based on the transformation mechanism of bacteria and gene regulation. The bacteria used for the experiment was Escherichia coli and the genes introduces for the transformation were: gfp and bla by a pGLO™ plasmid. After the insertion of the target genes and growing the bacteria on specialized LB media, it could be seen that the transformants were positive for the gene expression. The transformed E. coli on the media appeared fluorescent green under UV light.
The bacteria used in this experiment is Escherichia coli which is not naturally competent. E. coli is a gram negative rod shaped bacteria and a facultative anaerobe. This bacteria forms part of the bacterial flora in the human intestine tract. The competence of a bacteria is based on its ability to take up naked DNA from the environment and incorporated on theirs, transformation. Alteration in the permeability of the membranes allows DNA to cross the cell envelope of E. coli. Since the outer membrane of the E. coli is mostly negatively charged and the DNA molecule also has a negative charge, then the addition of CaCl2 will neutralize the interaction so that the naked DNA molecule can enter the cell. (Microbe Library web) Another important factor on the competence of the bacteria is a procedure of alternating temperature between ice bucket and heat shocks. By the combination of this two procedures E. coli becomes competent. This procedure was first reported by Mandel and Higa. (Singh 562) Even though it works it is only believed that CaCl2 helps DNA absorption to cell surface and the heat-shock step facilitates penetration of absorbed DNA into cell. (Panja 411) The main purpose of this experiment is to transform the bacteria to make it resistant to the antibiotic ampicillin. A secondary transformation is being made, and is to make the bacteria seem fluoresce green. The reason why the bacteria will fluoresce is because the gfp gene is being inserted under an ara promoter. The gfp gene encodes for the Green Fluorescent Protein (GFP). The genes under the ara promoter will be expressed when the bacteria is in presence of the sugar Arabinose. When the transformed E. coli is in presence of Arabinose, the gfp will make the GFP and when the bacteria is placed under UV light it will fluoresce green. The gfp gene was found and extracted from a jellyfish, Aequorea victoria, and is being used as a visible reporter for gene expression. (Garcia-Cayuela 172) To introduce the gfp into the bacterial cell it was needed to be by a plasmid, as well as the gene to make the E. coli resistant to ampicillin, bla gene. The bla gene encodes for the protein beta lactamase which breaks down the β-lactam ring in the structure of the ampicillin, therefore making it resistant to the antibiotic. Like already said to introduce this two genes to the E. coli it must be done through a plasmid. Both genes were introduced by the same one. In this case the one that was used was a pGLO™ plasmid. This is an engineered plasmid used as a vector to create genetically modified bacteria. This plasmid contains three specific genes: bla, gfp and araC. The ¬araC is a promoter region that regulates the expression of the gfp only under the presence of arabinose sugar. Materials and Methods
In this experiment a pGLO™ transformation kit was used. First we needed two eppie tubes, one pGLO positive and the other pGLO negative. This two eppies were then moved to an ice bucket. During, one loopful of the pGLO plasmid was transfer to the pGLO+ tube. The other tube will be the pGLO-, the Escherichia coli without the plasmid. The two tubes were moved into an ice bucket and left there for 10 minutes. Then the tubes were put into a 42º C water bath for 50 seconds and after back to the ice bucket for 2 minutes more. After the two minutes...