Transformation of E. coli
Table of Contents
Growth Stage issue
Advantages of Klenow
Advantages of CaCl2
Advantages of Log Phase
Escherichia coli (E. coli) are bacteria that contain DNA and plasmids. Plasmids express genes that are encoded in the vector. Genes are the basis for bacterial transformations. E. coli are the most commonly transformed bacteria. Transforming bacteria uses DNA (gene) of another organism to cause a heritable change. Heritable change is the manufacturing of different proteins by the addition of a gene. These proteins can now permanently change the phenotype of the cell. Changing the phenotype expresses the nature of the gene extracted from the original organism. A transformation phenotype is expressed in the form of color or resistance.
Plasmids are small, circular pieces of DNA that are typically 200 to 10,000 base pairs. A base pair is one purine in one strand linked by hydrogen bonds to a pyrimidine. Multiple purines and pyrimidines are the components of a gene. Genes contain important information for the growth of the bacteria. The genes making up the plasmid contain a selectable marker (a gene that encodes for antibiotic resistance), an origin of replication (which is used by the DNA polymerase in the bacteria as the starting point to make a copy of the plasmid), and a multiple cloning site (for inserts of genes). The cell utilizes these genetic components when being transformed. Transformation consists of multiple steps of insertion and performing chemically balanced reactions.
(red represents gene and blue represents vector)
The artificial transformation process consists of:
1. Gene and vector ligation to make plasmid
2. Cells that are made competent
3. Plasmid inserted into cell on LB+AMP (broth + ampacilin) plate 4. Cell reading and expressing the plasmid
5. Cell becoming transformed
6. Cell growing into colonies
Artificial transformation relocates genetic information between organisms. Transformation occurs naturally between organisms to exchange genes. DNA is transferred during the life and death of a cell. Transformations make cells vary in genotype and phenotype. Transforming many genes into bacteria gives the cell resistance to environmental factors. The multiple gene transformation causes super bugs. Super bugs are highly antibiotic resistant bacteria. Highly antibiotic resistant bacteria contain twenty or more environmental resistance genes.
Transformation makes bacteria stronger and more “fit” to produce colonies. This phenomenon is spontaneous in nature or could be induced in a laboratory. DNA transformation under laboratory conditions has a one percent chance of naturally taking up the gene. Competency is the potential to allow a foreign gene to enter a cell. Before bacteria can be artificially transformed, they must become competent—able to take up DNA molecules. The DNA molecules are water-soluble and cell membranes are made of a very insoluble lipid bilayer. This bilayer makes DNA unable to enter the cell; therefore, artificial transformation cannot occur spontaneously.
Freeze and thaw technique is a method of creating competent cells. Freeze and thaw is the repetitive cycle of freezing the cell and heating it up. Freeze and thaw brakes the membrane. Breaking the membrane stimulates the gene to enter the cell. When the gene enters the cell, it is inserted into a plasmid (vector) within the cell. To make a good gene that can be inserted, the promoter and gene length have to be specialized. To be transformed, the E. coli vector must have a promoter to cause a chain reaction. The chain reaction produced by the promoter depends on the length of the gene. If the gene length...
Please join StudyMode to read the full document