RECOMBINANT DNA BACTERIAL TRANSFORMATION 1 COMPETENT CELLS Competent

RECOMBINANT DNA BACTERIAL TRANSFORMATION 1

COMPETENT CELLS Competent cells are bacterial cells that possess more easily altered cell walls by which foreign DNA can be passed through easily. Most types of cells cannot take up DNA efficiently unless they have been exposed to special chemical or electrical treatments to make them competent. 2

The standard method for making the bacteria permeable to DNA involves treatment with calcium ions. Electroporation is technique in which an electrical field is applied to cells in order to increase the permeability of the cell, allowing membrane, DNA to be introduced into the cell. However, some types of bacteria are naturally transformable, which means they can take up DNA from their environment without requiring special treatment. 3

Objective To be able know how cells are made competent which is the primary step for transformation 4

COMPETENCE S DISTINGUISHED INTO NATURAL COMPETENCE AND INDUCED OR ARTIFICIAL COMPETENCE. 1. Natural competence Bacteria are able to take up DNA from their environment by different ways such conjugation and transduction. In transformation the DNA is directly entered to the cell. 5

Transduction is injection of foreign DNA by a bacteriophage virus into the host bacterium. Conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. 6

2. Artificial Competence Can be induced in laboratory procedure by which cells are made permeable to DNA, with conditions that do not normally occur in nature. Methods for preparing the competent cells derive from the work of Mandel and Higa who developed a simple treatment based on soaking the cells in cold Ca. Cl 2. 7

The surface of bacteria such as E. coli is negatively due to phospholipids and lipopolysaccharides on its cell surface, and the DNA is also negatively charged. One function of the divalent cation therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface. 8

9

Rapidly growing cells are made competent more easily than cells in other Growth stages. So it is necessary to brought cells into log phase before the procedure is begun. The cells in rapid growth (log phase) are living, healthy, and actively metabolizing. 10

Electroporation is technique in which an electrical field (create holes in the cell membrane) is applied to cells in order to increase the permeability of the cell, allowing membrane, DNA to be introduced into the cell. 11

12

PRINCIPLE As DNA is negatively charged, normally it cannot pass through the cell membrane of bacteria. Hence, in order to make bacteria capable of internalizing the genetic material, they must be made competent to take up the DNA. This can be achieved by calcium chloride solution that prepares the cell membrane to become permeable to plasmid DNA 13

PROTOCOL 1. DAY 1: Inoculate LB medium with bacterial cells and incubate at 37°C. for 24 hrs. 2. DAY 2: Centrifuge the tubes of over-nigh cultured cells 4000 rpm for 10 min at 4°C. 3. Remove the media, resuspend the cell pellet with ice cold 0. 1 M cacl 2. 4. Incubate on ice for 30 min. 14

5. Centrifuge at 4000 rpm for 10 min at 4°C. 6. Remove the media, resuspend the cell pellet with ice cold 0. 1 M Mgcl 2 -cacl 2. 7. Incubate on ice for 20 min. 8. Add ice-cold 80% glycerol and swirl to mix. 9. Freeze 100 μL aliquot in liquid nitrogen. 10. Store in -80 °c 15