competent cell là gì, chuẩn bị competent cells như thế nào?


Principle:

 (Adapted from http://amrita.vlab.co.in/?sub=3&brch=77&sim=702&cnt=2)
Competent cells are ready to use 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 . The standard method for making the bacteria permeable to DNA involves treatment with calcium ions. Brief exposure of cells to an electric field also allows the bacteria to take up DNA and this process is called as electroporation . However, some types of bacteria are naturally transformable, which means they can take up DNA from their environment without requiring special treatment. The exact mechanisms involved in artificial competence are not yet known well. ln CaCl2method, the competency can be obtained by creating pores in bacterial cells by suspending them in a solution containing high concentration of calcium. DNA can then forced in to the Host cell by heat shock treatment at 42oC for the process of transformation.

 

 

Competence is distinguished into natural competence, a genetically specified ability of bacteria that is thought to occur under natural conditions as well as in the laboratory, and induced or artificial competence, arising when cells in laboratory cultures are treated to make them transiently permeable to DNA.

 

1. Natural Competence

 

Bacteria are able to take up DNA from their environment by three ways; conjugation, transformation, and transduction.  In transformation the DNA is directly entered to the cell.  Uptake of transforming DNA  requires the recipient cells to be in a specialized physiological state called competent state. Natural competence was first discovered by Frederich Griffith in 1928. It is highly regulated in bacteria, and the factors involved in competence vary among genera. The competence proteins  produced  have some homology but differ in the Gram negative and the Gram positive bacteria.  Once the DNA has been brought into the cell’s cytoplasm, it may be degraded by the nuclease enzymes, or, if it is very similar to the cells own DNA, the DNA repairing enzymes may recombine it with the chromosome.

 

2. Artificial Competence and Transformation

 

Artificial competence is not encoded in the cell’s genes. Instead it is a laboratory procedure by which cells are  made permeable to DNA, with  conditions that do not normally occur in nature. This procedure is comparatively easy and simple, and can be used in the genetic engineering of bacteria but in general transformation efficiency is low.  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 CaCl2 . There are two main methods for the preparation of competent cells .They are Calcium chloride method and Electroporation.

 

Overview of competence and heat shock

 

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. Competent cells are readily available in commercial markets.

 

 

Materials Required:-

 

  •  LB broth
  •  Culture plates
  •  Ice cold CaCl2.2H2O  (1  M)
  •  Ice cold MgCl2 CaCl2  solution
  •  Shaking incubator
  •  Vortex mixer
  •  Centrifuge
  •  Water  bath
  •  Inoculation loop
  •  Microfuge tubes
  •  Polypropylene  tubes
  •  Micro pipettes and tips

 

Procedure:-

 

  1. Pick a single bacterial colony  from a culture plate which is incubated for 16-20 hours at 37°C. Transfer the colony into 100 ml LB broth a 1-liter flask. Incubate the culture for 3 hours at 37°C with vigorous agitation, monitoring the growth of the culture. As a guideline, 1 OD600 of a culture of E. coli strain DH 5 alpha contains approx. 109 bacteria/ml.
  2. Transfer the bacterial cells to sterile, disposable, ice-cold 50ml polypropylene tubes. Cool the cultures to 0°C by storing them on ice for 10 minutes.
  3. Recover the cells by centrifugation at 2700g  at 4°C for 10 minutes .
  4. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away.
  5. Resuspend each pellet by swirling or gentle vortexing in 30 ml of ice-cold MgCl2-CaCl2 solution.
  6. Recover the cells by centrifugation at 2700g  at 4°C for 10 minutes .
  7. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away.
  8. Resuspend the pellet in  2 ml of ice-cold 0.1 M CaCl2 (or TFB) by gentle vortexing for each 50ml of original culture. Standard TFB may be used instead of CaCl2 for most strains of E. coli.
  9. At this point, either use the cells directly for transformation or dispense into aliquots and freeze at -70°C.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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