Transformation of Escherichia Coli (E.Coli) with Plasmid Pglo

Topics: DNA, Bacteria, Molecular biology Pages: 4 (1341 words) Published: November 1, 2011
Bacterial transformation is the permanent alteration of a bacterial cell genotype as a result of its uptake and incorporation of foreign DNA fragments from external medium (Anthony et al, 2008). In addition to chromosome, bacterial cells often contain extrachromosomal DNA called plasmids which are capable of autonomic replication and antibiotic resistance (Dale & Simon, 2010). Plasmids can transport foreign DNA into host or other bacterial cells hence they are known as vectors. The resultant DNA of the transportation is called recombinant DNA (Mader, 2010). During transformation, bacterial cells that receive foreign DNA are called transformants (Pierce, 2008). Bacterial cells which are capable of taken up foreign DNA are said to be competent, incompetent cells can be made competent by treatment with calcium chloride (Brown, 2006). Bacterial transformation has a lot of uses such as mapping bacterial chromosomes (Anthony et al, 2008). This experiment is aimed at exploring the transformation of E.coli bacteria with a genetically engineered DNA called pGLO plasmids. The pGLO plasmids are genetically engineered to code for a number of genes including the gene for Green Fluorescent Protein (GFP). The GFP, isolated from the jellyfish Aequorea victorea, glows in the presence of U.V light (Sanders and Jackson, 2009). Materials and Methods

A 250µl of a calcium chloride transformation solution ‘TS’ was added to each of two different tubes of Escherichia coli HB101 kept in an ice bucket. The tubes were marked as ‘pGLO’ and ‘control’ respectively. A 10µl of pGLO DNA sample was added to the tube marked ‘pGLO’. A 10µl of sterile water was added to the ‘control’ tube; both tubes were placed on ice for ten minutes, after which, they were incubated at 42°C for two minutes. The tubes were placed back on ice immediately for two minutes. A 250µl of LB broth was then added to each tube; both tubes were left at room temperature for ten minutes....
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