Lab Process-Transformation

In molecular biology, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacteria must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

Lab methods--Start with bacteria--say E Coli---Make Agar plates-- Label very carefully--usually Selective  (With antibiotic) and non-selective (no antibiotic)  So label S and NS  ----

1. Initial growth

2. Make Cells Competent

3. Let cells recover

1.  So start with making the agar plates and then doing the "Z" streak with the basic bacteria.---

2. Then they have to be made chemically competent. This involves the transformation buffer.  The bacterial cells that can take up the foreign DNA from the surroundings by a process called transformation are known as competent cells. The cells might express the acquired genetic information after transformation. The process is largely used to introduce recombinant plasmid DNA into competent bacterial cells. This process does not require a donor cell but only a DNA in the surrounding environment.  Competent cells have altered cell walls that allow the DNA to easily pass through it. Some cells need to be exposed to some chemical or electrical treatments to make them competent. Treatment with calcium ions is the standard method for the preparation of these cells. Bacterial cells can also take up DNA through a process called electroporation.  It requires usually about 15 minutes at 4 degrees cent followed by  a heat shock of 42 degress cent for 90 secs and then 3 monutes back at 4 degrees. Then to Recovery stage

Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or DNA to be introduced into the cell (also called electrotransfer). In microbiology, the process of electroporation is often used to transform bacteria, yeast, or plant protoplasts by introducing new coding DNA. If bacteria and plasmids are mixed together, the plasmids can be transferred into the bacteria after electroporation, though depending on what is being transferred cell-penetrating peptides or CellSqueeze could also be used. Electroporation works by passing thousands of volts across a distance of one to two millimeters of suspended cells in an electroporation cuvette (1.0 – 1.5 kV, 250 – 750 V/cm). Afterwards, the cells have to be handled carefully until they have had a chance to divide, producing new cells that contain reproduced plasmids. This process is approximately ten times more effective than chemical transformation.

3. Recovery Phase and Transformation   You add recovery media to the now competent cell mixture. and treat at 37 degress for almost 1 Hour.  Now cells are transformed --you just need to culture them for 24-72 hours. Don't rush this one.  Recovery media is usually made up---This nutrient-rich microbial broth contains peptides, amino acids, water-soluble vitamins, and glucose in a low-salt formulation.

Components of recovery media--typical for E coli  often called S.O.C. medium --this works a little beter then LB Broth (Lennox L Broth) 

20 g/L Tryptone

5 g/L Yeast Extract

4.8 g/L MgSO4

3.603 g/L dextrose

0.5g/L NaCl

0.186 g/L KCl

Lab Process-Transformation

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