Genetic transformation is the process by which DNA is moved into an organism so that its genotype is altered. Experiments to observe this can be really expensive and complicated, but gene transfer in E.Coli bacteria are simple. In this specific lab, bacteria is transformed with a gene that codes for GFP (Green Fluorescent Protein). This allows the bacteria with this gene to glow a green color when it is exposed to ultraviolet light. Also, plasmid DNA which contains genes that help it survive can also be observed.
pGLO resists to the antibiotic that is used (ampicillin). This means that those bacteria that resist are expected to continue to grow and form colonies. The gene for GFP can be switched on in those cells that have been transformed, by adding sugar to the nutrients. Those that do not have the sugar will appear white on LB/Amp plates.
The purpose of this lab was to get a clear understanding of how gene transformation happens and the impact it has on bacteria.
The hypothesis for this lab was that if bacteria was put in the LB- plasmid plate (no plasmid) and in the LB/ Amp – plasmid plate (AMP present and no plasmid) there would be no growth. If bacteria was put in the LB + plasmid plate ( has plasmid) and in the LB/Amp + plasmid plate ( has plasmid and will defeat the AMP that is also there), there would be bacteria growth. Some of the questions we intend to answer by the end of this lab are: What does the phenotype of the transformed colonies tell you? What would one plate you first inspect to conclude that the transformation occurred successfully? What factors might influence transformation efficiency?
1). Mark one sterile 15 mL tube “+ plasmid” (plasmid added)
Mark another “- plasmid” (plasmid not added)
2). Use a sterile transfer pipet to add 250 ?L of ice-cold calcium chloride to each tube.
3). Place both tubes on ice.
4). Use a sterile plastic inoculating loop to transfer isolated colonies of E.Coli from …