AIM: To create competent E.coli cells by using the chemical method (calcium chloride treatment) To transform the competent bacterial cells using plasmid DNA and evaluate the transformation efficiency.
PRINCIPLE:
Competent cells are pre-prepared bacterial cells that possess altered cell walls through which foreign DNA can enter the cell easily. Most cells cannot take up DNA efficiently unless they have been treated with chemicals or exposed to electrical treatments in order to make them competent. The standard method used for this purpose involves treatment with calcium ions. Exposure to an electric field, for a brief duration, also allows DNA take up by the bacteria. This process is called electroporation. Some bacterial cells are naturally …show more content…
It is an easy process and has been used in genetic engineering of bacteria but in general transformation efficiency is low. Methods for preparing competent cells have been derived from the work of Morton Mandel and Akiko Higa, who had developed a simple treatment based on soaking the cells in cold calcium chloride. Rapidly growing cells are made competent more easily than cells in other growth stages. So it is necessary to bring the cells to log phase before the procedure is done.
In Calcium Chloride method, when the cells are treated with calcium chloride, the divalent cation Ca2+ neutralizes the negative charge on the cell membrane and increases the permeability. When this is followed by heat shock, the foreign DNA that is kept in close proximity with the cell is taken up by the …show more content…
This results in the acquisition of new genetic characteristics by the transformed cell. The two major parameters involved in efficient transformation of a bacterial cell is the method used to induce competence and the genetic constitution of the host being transformed. Competent cells are capable of taking up foreign DNA and expressing it as functional proteins. If a bacterium is said to be competent, it has to maintain a physiological state in which it can take up the donor DNA. Calcium chloride treatment is one of the best methods for the preparation of competent cells. Through this bacteria can transfer advantageous characteristics, such as antibiotic resistance. Bacteria can take up DNA from the environment in the form of plasmid. Most plasmids are double stranded circular DNA molecules and can exist at very high copy numbers within a single bacterial cell. Many naturally occurring plasmids carry an antibiotic resistant gene referred to as a
PRINCIPLE:
Competent cells are pre-prepared bacterial cells that possess altered cell walls through which foreign DNA can enter the cell easily. Most cells cannot take up DNA efficiently unless they have been treated with chemicals or exposed to electrical treatments in order to make them competent. The standard method used for this purpose involves treatment with calcium ions. Exposure to an electric field, for a brief duration, also allows DNA take up by the bacteria. This process is called electroporation. Some bacterial cells are naturally …show more content…
It is an easy process and has been used in genetic engineering of bacteria but in general transformation efficiency is low. Methods for preparing competent cells have been derived from the work of Morton Mandel and Akiko Higa, who had developed a simple treatment based on soaking the cells in cold calcium chloride. Rapidly growing cells are made competent more easily than cells in other growth stages. So it is necessary to bring the cells to log phase before the procedure is done.
In Calcium Chloride method, when the cells are treated with calcium chloride, the divalent cation Ca2+ neutralizes the negative charge on the cell membrane and increases the permeability. When this is followed by heat shock, the foreign DNA that is kept in close proximity with the cell is taken up by the …show more content…
This results in the acquisition of new genetic characteristics by the transformed cell. The two major parameters involved in efficient transformation of a bacterial cell is the method used to induce competence and the genetic constitution of the host being transformed. Competent cells are capable of taking up foreign DNA and expressing it as functional proteins. If a bacterium is said to be competent, it has to maintain a physiological state in which it can take up the donor DNA. Calcium chloride treatment is one of the best methods for the preparation of competent cells. Through this bacteria can transfer advantageous characteristics, such as antibiotic resistance. Bacteria can take up DNA from the environment in the form of plasmid. Most plasmids are double stranded circular DNA molecules and can exist at very high copy numbers within a single bacterial cell. Many naturally occurring plasmids carry an antibiotic resistant gene referred to as a