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

By Jogwunte Nov 01, 2011 1341 Words
Introduction
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. A 200µl of the ‘pGLO’ solution was spread on an LB agar plate containing ampicillin; the same quantity of the ‘pGLO’ solution was also spread on another LB agar plate containing ampicillin and arabinose. Also, a 200µl of the ‘control’ solution was spread on a third LB agar plate containing ampicillin. Seven sterile tubes numbered as -1 to -7 were set up; each contained 900µl of sterile LB broth. A 100µl of the ‘Control’ solution was added to tube -1; a 100µl of tube -1 was removed and added to tube -2. The same procedure of removing 100µl from successive tube and transferring to progressive tube was repeated for all tubes. A 100µl of the serial solution from -3 to the -7 were spread on separate LB agar plate and the whole plates were incubated at 37°C for one week.

Results
The ‘control’ was spread on LB+AMP plate; the remaining sample was used in setting up serial dilutions. The results after a week of incubation are shown in table 1. Table 1. The number of bacterial colonies derived from the serial dilution of the ‘control’ tube Dilution series Number of colonies|

-3 TMTC-4 TMTC-5 TMTC-6 TMTC-7 44|

The -7 dilution series has the least amount of colonies, while -3 to -6 have too many colonies to count; the cells in the ‘control’ tube where not resistant to ampicillin (table 1). Also, ‘pGLO’ was spread on LB+AMP and LB+AMP+ARABINOSE plates. The results after a week of incubation are shown in table 2. Table 2. The number of bacterial cells transformed, total number of cells and the transformation frequency Number of cells transformed by the pGLO plasmid| 35 Cells per ml| Total number of cells | 4.4x109 Cell per ml|

The frequency of transformation| 7.95 x10-9 |
Transformation frequency = Number of cells transformed by the pGLO plasmid / total cells = 35 Cells per ml/4.4x x109 Cell per ml

= 7.95x10-9 = 1/ (125786163). Table 2 showed that 35 cells were transformed out of a total of 4.4x x109 cells. The transformation frequency of 7.95 x10-9 means that 1 in every 125786163 cells was transformed. Furthermore, LB+AMP and LB+AMP+ARABINOSE plates were spread with ‘pGLO’ and incubated for a week after which they were observed under long U.V light. The results of the observation are shown in table 3. Table 3. The observation of LB+AMP and LB+AMP+ARABINOSE plates respectively under U.V light Plate Fluorescence under long U.V light (yes or no)| LB+AMPLB+AMP+ARABINOSE | No Yes| The pGLO+LB+AMP did not fluoresce, while the pGLO+LB+AMP+ARABINOSE glowed green (table 3). Discussion

There was massive growth of colonies on the five serial dilution plates (table 1). These plates contained only control+LB hence the growth is that of living bacteria. The cells were untransformed because they were not mixed with ‘pGLO’. However, there was no growth on the control+AMP+LB plate; this is because the plate did not have any antibiotic resistance hence cells were killed by the ampicillin. Meanwhile, colonies on pGLO+LB+AMP+ARABINOSE and pGLO+LB+AMP plates were transformed; the evidence was that, the former glowed green while the later did not glow on exposure to long U.V light (table 2&3). A transformation frequency of 7.95 x10-9 was obtained, which means 1 in every 125786163 cells was transformed (table 2). The low transformation frequency can be associated with the use of calcium chloride; because transformation frequency decreases rapidly with size of DNA when cells are treated with calcium chloride than when method such as electroporation (subjecting cells to a high-voltage electric field) are used (Primrose and Twyman, 2006). Also, bacterial cells possessed a limited number of receptor sites on their surface hence only competent cells in a particular physiologic state can bind and undergo transformation (William et al, 2009). The recombinant plasmid called pGLO codes for three genes which are bla, ara C and GFP. The bla gene codes for the enzyme beta-lactamase that allows bacteria to survive in an environment of ampicillin; ara C produces a protein that will turn on GFP gene in the presence of arabinose, while GFP gene when turned on produces Green Fluorescent Protein (GFP) that glows in the presence of U.V light (Mosher, 2002). The bla gene justified why the cells in both plates survived in the presence of ampicillin, while the ara C and GFP genes showed why only the plate with arabinose produced light. Only GFP was obtained in the presence of arabinose because the genes which code for arabinose catabolism, ara B, A and D, have been replaced with GFP gene; ara C promotes the binding of RNA polymerase and GFP gene in the presence of arabinose, the complex is then translated into GFP protein that glows in the presence of light (William et al, 2009; Mosher, 2002). References

Brown T. A., (2006). Gene Cloning and DNA Analysis An Introduction, 5th ed., Blackwell Publishing, UK, pp. 90 Dale J. W. and Park. S. F., (2010). Molecular Genetics of Bacteria, 5th ed., John Wiley and sons, Ltd. UK, pp. 148 & 354 Griffiths A. J. F., Wessler S. R., Lewontin R. C and Carroll S. B., (2008). Introduction to Genetic Analysis, 9th ed., W. H. Freeman and Company, USA; pp. 198 and 199 Klug W. S., Cumming M. R., Spenser C. A. and Palladino M. R., (2009). Concept of genetics. 9th ed., Pearson Education, Inc., NY, pp. 154, 448 and 449 Mader S. S., (2010). Biology; McGraw-Hill, 10th ed., pp. 64, 250 and 363 Mosher R. H. Using pGLO to Demonstrate the Effects of Catabolite Repression on Gene Expression in Escherichia coli.2002, 28(3), 18 Pierce A. B., (2008). Genetic: A Conceptual Approach, 3th ed., W. H. Freeman and Company, USA; pp. 214 Primrose S. B. And Twyman R. M (2006). Principle of gene manipulation and genomics, 7th ed., Wiley-Blackwell, USA; pp. 25 Sanders J. K. M. and Jackson S. E. Chem. Soc. Rev., 2009, 38, 2821-2822

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