- It Is Very Important For The Success Of Your Artificial Transformation That You Use The Right Concentration Of The Cacl 1 (15.44 KiB) Viewed 35 times
- It Is Very Important For The Success Of Your Artificial Transformation That You Use The Right Concentration Of The Cacl 2 (61.44 KiB) Viewed 35 times
- It Is Very Important For The Success Of Your Artificial Transformation That You Use The Right Concentration Of The Cacl 3 (61.44 KiB) Viewed 35 times
Bacterial Transformation-Part B As mentioned in Chapter 19, not all bacteria are naturally competent (naturally trans- formable). However, methods have been developed to produce competency in naturally incompetent bacteria and to transform these bacteria artificially. One method is the treat- ment of bacterial cells with an ice-cold calcium chloride (CaCl,) solution, which enables the cells to uptake DNA from their environment. It is not the cell wall that prevents the passage of intact DNA; the true barrier is the cell membrane. The drastic treatment with CaCl, allows foreign DNA to pass through the membrane and into the cell. The exact mechanism is still not known yet. What is known is that the CaCl, at a concentration of 100 mM makes the cell membrane more permeable. Another membrane-altering method is electroporation where cells are subjected to high voltage electric impulses that desta- bilize the cell membrane, resulting in increased permeability and enabling DNA to pass into the cells. In this lab you will use a plasmid to transform the bacterial cells. Plasmids are small circular pieces of extrachromosomal, double-stranded DNA that can be found in a variety of different bacteria species. They replicate independently of the bacterial chromosome and range in size from a few thousand base pairs (bp) to more than 100 kilo base pairs (kb). Plasmid cloning vectors are plasmids that are used for transformation experiments and, therefore, have been genetically engineered. For example, they contain an antibiotic resistance gene and a region in which exogenous DNA fragments can be inserted. In this lab period you will make E. coli cells arti- ficially competent by using the CaCl, method (see procedures). The plasmid that is used for the trans- formation of E. coli is the pGLO plasmid (see Fig. 20.1), which contains several reporter genes, most notably for the Green Fluorescent Protein (GFP) and the beta-lactamase antibiotic resistance gene (bla). The real-life source of the GFP gene is the bioluminescent jellyfish. Green Fluorescent Protein ori CHAPTER 2 pGLO 5400 bp bla arac GFP Figure 20.1: PGLO plasmid.